Production method for poly(amino acid)

ABSTRACT

The present invention relates to a graft copolymer of a poly(amino acid) or a salt thereof and a hydrophobic primary amine compound or a salt thereof (e.g., a graft copolymer (γ-PGA-PAE) of poly(γ-glutamic acid) (γ-PGA) and phenylalanine ethyl ester (PAE)), an ionized graft copolymer of a poly(amino acid) or a salt thereof and a hydrophobic primary amine compound or a salt thereof, nanoparticles containing the ionized graft copolymer, and a production method thereof. The nanoparticles acquired in this way are useful as an adjuvant for producing a vaccine.

This is a Continuation Application of U.S. patent application Ser. No.14/392,185, filed Dec. 23, 2015, an application claiming the benefitunder 35 U.S.C. 371 as a national stage of International Application No.PCT/JP2014/066870, filed Jun. 25, 2014 (the contents of which are herebyincorporated by reference in its entirety), which itself claims thebenefit of Japanese Application No. 2013-133917, filed Jun. 26, 2013.

TECHNICAL FIELD

The present invention relates to a graft copolymer of a poly(amino acid)or a salt thereof and a hydrophobic primary amine compound or a saltthereof [e.g., a graft copolymer (γ-PGA-PAE) of poly(γ-glutamic acid)(γ-PGA) and phenylalanine ethyl ester (PAE)], which is useful as anadjuvant for producing a vaccine, an ionized graft copolymer of apoly(amino acid) or a salt thereof and a hydrophobic primary aminecompound or a salt thereof, nanoparticles containing the ionized graftcopolymer, and a production method thereof.

BACKGROUND ART

Studies are recently conducted for utilizing nanoparticles as a drugcarrier (see Patent Documents 1 and 2).

Patent Document 3 discloses that a poly(amino acid) (e.g., γ-PGA) or agraft copolymer of a poly(amino acid) and a hydrophobic primary aminecompound or a salt thereof (e.g., γ-PGA-PAE) promotes differentiationand maturity of dendritic cells, i.e., acts as an adjuvant and that theadjuvant action is enhanced by formation of nanoparticles of the graftcopolymer.

Patent Document 3 discloses in Example 1 a production method ofγ-PGA-PAE using a salting-out method for desalting after reaction ofγ-PGA with carbodiimide hydrochloride and PAE, and a method of producingnanoparticles from γ-PGA-PAE.

PRIOR ART DOCUMENT Patent Documents

Patent Document 1: Japanese Laid-Open Patent Publication No. 6-92870

Patent Document 2: Japanese Laid-Open Patent Publication No. 6-256220

Patent Document 3: WO 2006/112477

SUMMARY OF THE INVENTION Problem to be Solved by the Invention

What is required is a method of producing a graft copolymer of apoly(amino acid) or a salt thereof and a hydrophobic primary aminecompound or a salt thereof useful as an adjuvant for a vaccine in alarge amount in a short time.

Means for Solving Problem

As a result of intensive studies for solving the problem, the inventorshave found a method of producing a free form of a graft copolymer of apoly(amino acid) or a salt thereof and a hydrophobic primary aminecompound or a salt thereof in a short period of time at a high yield byusing a method of precipitation by acid addition for isolation of thegraft copolymer.

The inventors also have found a method of acquiring nanoparticles withhigher efficiency and higher reproducibility than those of theconventional methods by partially or entirely ionizing the free form ofthe graft copolymer of a poly(amino acid) or a salt thereof and ahydrophobic primary amine compound or a salt thereof. This method alsoenables a scale-down of manufacturing equipment and a reduction of thewaste. Additionally, even when it is conventionally difficult to formnanoparticles from the free form of the graft copolymer of a poly(aminoacid) or a salt thereof and a hydrophobic primary amine compound or asalt thereof, the partial or entire ionization facilitates the formationof nanoparticles.

Therefore, the present invention relates to:

[1] a production method of a graft copolymer of a poly(amino acid)selected from the group consisting of poly(γ-glutamic acid),poly(α-glutamic acid), and poly(aspartic acid) or a salt thereof and ahydrophobic primary amine compound represented by Formula (I): A-NH₂[wherein A denotes a hydrophobic moiety] or a salt thereof, the methodcomprising the steps of:

(1) acquiring a graft copolymer by condensation of the poly(amino acid)or a salt thereof with the hydrophobic primary amine compoundrepresented by formula (I) or a salt thereof; and

(2) isolating the graft copolymer by allowing an acid to act on thegraft copolymer acquired at step (1);

[2] the production method according to [1], wherein at step (2), theacid is allowed to act on the graft copolymer at a temperature of 0 to80° C.;

[3] the production method according to [1], wherein the poly(amino acid)is poly(γ-glutamic acid);

[4] the production method according to [1], wherein the hydrophobicprimary amine compound is an α-amino acid derivative;

[5] the production method according to [4], wherein the α-amino acidderivative is a phenylalanine derivative;

[6] the production method according to [5], wherein the phenylalaninederivative is phenylalanine ethyl ester;

[7] a graft copolymer of a poly(amino acid) selected from the groupconsisting of poly(γ-glutamic acid), poly(α-glutamic acid), andpoly(aspartic acid) or a salt thereof and a hydrophobic primary aminecompound represented by Formula (I): A-NH₂ [wherein A denotes ahydrophobic moiety] or a salt thereof, the graft copolymer beingproduced with the method according to [1];

[8] a production method of an ionized graft copolymer of a poly(aminoacid) selected from the group consisting of poly(γ-glutamic acid),poly(α-glutamic acid), and poly(aspartic acid) or a salt thereof and ahydrophobic primary amine compound represented by Formula (I): A-NH₂[wherein A denotes a hydrophobic moiety] or a salt thereof, the methodcomprising the step of:

ionizing the graft copolymer by allowing a hydroxide of alkali metal, acarbonate of alkali metal, a hydrogencarbonate of alkali metal, aphosphate alkali metal, a monohydrogen phosphate of alkali metal, adihydrogen phosphate of alkali metal, an organic acid salt of alkalimetal, or an acidic amino-acid salt of alkali metal to act on the graftcopolymer of the poly(amino acid) or a salt thereof and the hydrophobicprimary amine compound or a salt thereof;

[9] An ionized graft copolymer represented by Formula (II):

[wherein M is alkali metal, A is a hydrophobic moiety, and n is aninteger from 10 to 100,000; diagonal lines intervening three monomerunits represent that the monomer units are arranged in irregular order;and x is a mole fraction of the monomer unit represented by Formula(III):

y is a mole fraction of the monomer unit represented by Formula (IV):

z is a mole fraction of the monomer unit represented by Formula (V):

andx, y, and z satisfy the following equations:0≤x<1;0<y<1;0<z<1; andx+y+z=1];  [Math. 1]

[10] the ionized graft copolymer according to [9], wherein the molefraction x of the monomer unit represented by Formula (III) is 0;

[11] the ionized graft copolymer according to [9], wherein n is 50 to10,000;

[12] the ionized graft copolymer according to [9], wherein the ionizedgraft copolymer has a hydrophobic parameter K of −15,000 to 0;

[13] a production method of nanoparticles containing an ionized graftcopolymer of a poly(amino acid) selected from the group consisting ofpoly(γ-glutamic acid), poly(α-glutamic acid), and poly(aspartic acid) ora salt thereof and a hydrophobic primary amine compound represented byFormula (I): A-NH₂ [wherein A denotes a hydrophobic moiety] or a saltthereof.

the method comprising the step of forming nanoparticles of the ionizedgraft copolymer produced with the production method according to [8];

[14] the production method of nanoparticles according to [13],comprising the steps of:

(1) condensing a poly(amino acid) selected from the group consisting ofpoly(γ-glutamic acid), poly(α-glutamic acid), and poly(aspartic acid) ora salt thereof and a hydrophobic primary amine compound represented byFormula (I): A-NH₂ [wherein A denotes a hydrophobic moiety] or a saltthereof;

(2) isolating a graft copolymer by allowing an acid to act on acondensate acquired at step (1);

(3) ionizing the graft copolymer by allowing a hydroxide of alkalimetal, a carbonate of alkali metal, a hydrogencarbonate of alkali metal,a phosphate of alkali metal, a monohydrogen phosphate of alkali metal, adihydrogen phosphate of alkali metal, an organic acid salt of alkalimetal, or an acidic amino-acid salt of alkali metal to act on the graftcopolymer isolated at step (2); and

(4) forming nanoparticles of the ionized graft copolymer acquired atstep (3);

[15] nanoparticles produced by the method according to [13], thenanoparticles containing an ionized graft copolymer of a poly(aminoacid) selected from the group consisting of poly(γ-glutamic acid),poly(α-glutamic acid), and poly(aspartic acid) or a salt thereof and ahydrophobic primary amine compound represented by Formula (I); A-NH₂[wherein A denotes a hydrophobic moiety] or a salt thereof;

[16] the nanoparticles according to [15], wherein the nanoparticles areused as an adjuvant; and

[17] a vaccine containing the nanoparticles according to [15].

Effect of the Invention

According to the present invention, a graft copolymer of a poly(aminoacid) or a salt thereof and a hydrophobic primary amine compound or asalt thereof may be acquired in a shorter time at a higher yield thanthose of the conventional methods.

According to the present invention, nanoparticles containing the graftcopolymer of a poly(amino acid) or a salt thereof and a hydrophobicprimary amine compound or a salt thereof may be acquired with highefficiency and good reproducibility.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing that nanoparticles of γ-PGA-PAE according tothe present invention have excellent performance as an adjuvant for avaccine.

MODES FOR CARRYING OUT THE INVENTION

A first aspect of the present invention relates to a graft copolymer ofa poly(amino acid) selected from the group consisting of poly(γ-glutamicacid), poly(α-glutamic acid), and poly(aspartic acid) or a salt thereofand a hydrophobic primary amine compound represented by Formula (I):A-NH₂ [wherein A denotes a hydrophobic moiety] or a salt thereof, and aproduction method thereof.

The production method of the graft copolymer according to the firstaspect of the present invention comprises the steps of:

(1) acquiring a graft copolymer by condensation of the poly(amino acid)or a salt thereof with the hydrophobic primary amine compoundrepresented by Formula (I) or a salt thereof; and

(2) isolating the graft copolymer by allowing an acid to act on thegraft copolymer acquired at step (1).

In this description, a “poly(amino acid)” means an amino acid chain madeup of a plurality of bonded amino acids.

Examples of amino acids making up a poly(amino acid) include glutamicacid (e.g., α-glutamic acid, γ-glutamic acid), aspartic acid, lysine,asparagine, arginine, and the like.

The constituent amino acids in the poly(amino acid) may be theL-isomers, the D-isomers, or the mixture thereof.

A bond between the constituent amino acids in the poly(amino acid) maybe a peptide bond, a bond other than a peptide bond, such as an esterbond and an ether bond, or a bond via a linker such as glutaraldehydeand diisocyanate, and is typically a peptide bond.

In a preferred embodiment, the poly(amino acid) has a number averagemolecular weight of 1 to 20,000 kDa, preferably 20 kDa to 3,000 kDa. Inthis description, a molecular weight may be a relative molecular weightor an absolute molecular weight. For example, the relative molecular,weight is a numerical value determined by a molecular weight measurementmethod using the following SEC-HPLC measurement: TSKgel α-M 300×7.8 mmI.D. (dual), 5 mM NaNO₃ DMSO:H₂O (9:1), 0.8 mL/minute, 40° C., RIdetector, standard: pullulan (Shodex). The absolute molecular weight isa numerical value determined by a molecular weight measurement methodusing the following SEC-HPLC conditions: TSKgel GMPWXL, 300×7.8 mm I.D.(dual), 0.1 M NaNO₃, 0.8 mL/minute, 40° C., simultaneous detection by RIdetector, viscometer, DLS detector, and SLS detector.

Specific examples of the poly(amino acid) include poly(γ-glutamic acid),poly(α-aspartic acid), poly(ε-lysine), poly(α-glutamic acid), andpoly(α-lysine) and, among them, poly(γ-glutamic acid), poly(α-glutamicacid), and poly(α-aspartic acid) are preferable. Most preferably, thepoly(amino acid) is poly(γ-glutamic acid).

Examples of the salt of the poly (amino acid) used in the presentinvention include, for example, a metal salt, an ammonium salt, a saltwith an organic base, inorganic acid a salt with, a salt with an organicacid, a salt with a basic or acidic amino acid, and the like. Preferredexamples of the metal salt include, for example, alkali metal salts suchas a sodium salt and a potassium salt; alkaline earth metal salts suchas a calcium salt, a magnesium salt, and a barium salt; aluminum salts,and the like. Preferred examples of the salt with an organic baseinclude, for example, salts with trimethylamine, triethylamine,pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine,triethanolamine, cyclohexylamine, dicyclohexylamine,N,N′-dibenzylethylenediamine, N-methylmorpholine,N,N-diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), andthe like. Preferred examples of the salt with an inorganic acid include,for example, salts with hydrochloric acid, hydrobromic acid, nitricacid, sulfuric acid, phosphoric acid, and the like. Preferred examplesof the salt with an organic acid include, for example, salts with formicacid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid,oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid,malic acid, methanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid, lactic acid, benzoic acid, and the like.Preferred examples of the salt with a basic amino acid include, forexample, salts with arginine, lysine, ornithine, and the like, andpreferred examples of the salt with an acidic amino acid include, forexample, salts with aspartic acid, glutamic acid, and the like. Amongthem, preferable salts are pharmaceutically acceptable salts including asodium salt, a potassium salt, and a lithium salt, and the sodium saltis particularly preferably.

In this description, in the hydrophobic primary amine compoundrepresented by Formula (I): A-NH₂ [wherein A denotes a hydrophobicmoiety], A- denoting the “hydrophobic moiety” means a derivative havingan aromatic ring group such as a benzene ring, a derivative having aC₃₋₈ carbon chain, a derivative having a C₈₋₂₂ linear fatty chain, aderivative represented by R¹—(CHR²)—, and a derivative represented byR³—(CR⁴R⁵)—(CR⁶R⁶)—.

Examples of the “derivative having an aromatic ring group” representedby the A- include, for example, C₆₋₁₄ aryl groups that may have asubstituent (e.g., phenyl, 1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl,9-anthryl), C₇₋₁₆ aralkyl groups that may have a substituent (e.g.,benzyl, phenethyl, naphthylmethyl, phenylpropyl), and aromaticheterocyclic groups that may have a substituent, and the like.

Examples of the aromatic heterocyclic groups of the “aromaticheterocyclic groups that may have a substituent” include, for example,5- to 14-membered (preferably, 5- to 10-membered) aromatic heterocyclicgroups containing 1 to 4 heteroatoms as annular atoms selected fromnitrogen atoms, sulfur atoms, and oxygen atoms in addition to carbonatoms.

Preferred examples of the “aromatic heterocyclic groups” include:

5- to 6-membered monocyclic aromatic heterocyclic groups such asthienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl,isothiazolyl, oxazolyl, isooxazolyl, pyridyl, pyrazinyl, pyrimidinyl,pyridazinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl,1,3,4-thiadiazolyl, triazolyl, tetrazolyl, and triazinyl; and

8- to 14-membered condensed polycyclic (preferably, bicyclic ortricyclic) aromatic heterocyclic groups such as benzothiophenyl,benzofuranyl, benzoimidazolyl, benzooxazolyl, benzoisooxazolyl,benzothiazolyl, benzoisothiazolyl, benzotriazolyl, imidazopyridinyl,thienopyridinyl, furopyridinyl, pyrrolopyridinyl, pyrazolopyridinyl,oxazolopyridinyl, thiazolopyridinyl, imidazopyrazinyl,imidazopyrimidinyl, thienopyrimidinyl, furopyrimidinyl,pyrrolopyrimidinyl, pyrazolopyrimidinyl, oxazolopyrimidinyl,thiazolopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thienyl,phenoxathiinyl, indolyl, isoindolyl, 1H-indazolyl, purinyl, isoquinolyl,quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl,cinnolinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl,phenazinyl, phenothiazinyl, and phenoxazinyl.

The number of substituents at substitutable positions of the “C₆₋₁₄ arylgroup that may have a substituent” represented by the A- is, forexample, 1 to 5, preferably 1 to 3. If the number of substituents is notless than 2, the substituents may be the same or different.

The substituents may be selected from C₁₋₆ alkyl groups that may behalogenated (e.g., methyl, chloromethyl, difluoromethyl,trichloromethyl, trifluoromethyl, ethyl, 2-bromoethyl,2,2,2-trifluoroethyl, tetrafluoroethyl, pentafluoroethyl, propyl,2,2-difluoropropyl, 3,3,3-trifluoropropyl, isopropyl, butyl,4,4,4-trifluorobutyl, isobutyl, sec-butyl, tert-butyl, pentyl,isopentyl, neopentyl, 5,5,5-trifluoropentyl, hexyl,6,6,6-trifluorohexyl), C₆₋₁₄ aryl groups (e.g., phenyl, 1-naphthyl,2-naphthyl, 1-anthryl, 2-anthryl, 9-anthryl), C₇₋₁₆ aralkyl groups(e.g., benzyl, phenethyl, naphthylmethyl, phenylpropyl), and thefollowing Substituent Group a.

[Substituent Group a]

-   (1) halogen atoms (e.g., fluorine, chlorine, bromine, iodine),-   (2) nitro groups,-   (3) cyano groups,-   (4) hydroxy groups,-   (5) C₁₋₆ alkoxy groups that may be halogenated (e.g., methoxy,    ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy,    tert-butoxy, pentyloxy, hexyloxy),-   (6) C₆₋₁₄ aryloxy groups (e.g., phenoxy, naphthoxy),-   (7) C₇₋₁₆ aralkyloxy groups (e.g., benzyloxy),-   (8) 5- to 14-membered aromatic heterocyclic oxy groups (e.g.,    pyridyloxy),-   (9) 3- to 14-membered non-aromatic heterocyclic oxy groups (e.g.,    morpholinyloxy, piperidinyloxy),-   (10) C₁₋₆ alkyl-carbonyloxy groups (e.g., acetoxy, propanoyloxy),-   (11) C₆₋₁₄ aryl-carbonyloxy groups (e.g., benzoyloxy,    1-naphthoyloxy, 2-naphthoyloxy),-   (12) C₁₋₆ alkoxy-carbonyloxy groups (e.g., methoxycarbonyloxy,    ethoxycarbonyloxy, propoxycarbonyloxy, butoxycarbonyloxy),-   (13) mono- or di-C₁₋₆ alkyl-carbamoyloxy groups (e.g.,    methylcarbamoyloxy, ethylcarbamoyloxy, dimethylcarbamoyloxy,    diethylcarbamoyloxy),-   (14) C₆₋₁₄ aryl-carbamoyloxy groups (e.g., phenylcarbamoyloxy,    naphthylcarbamoyloxy),-   (15) 5- to 14-membered aromatic heterocyclic carbonyloxy groups    (e.g., nicotinoyloxy),-   (16) 3- to 14-membered non-aromatic heterocyclic carbonyloxy groups    (e.g., morpholinylcarbonyloxy, piperidinylcarbonyloxy),-   (17) C₁₋₆ alkylsulfonyloxy groups that may be halogenated (e.g.,    methylsulfonyloxy, trifluoromethylsulfonyloxy),-   (18) C₆₋₁₄ arylsulfonyloxy groups that may be substituted with C₁₋₆    alkyl groups (e.g., phenylsulfonyloxy, toluenesulfonyloxy),-   (19) C₁₋₆ alkylthio groups that may be halogenated (e.g.,    methylthio, ethylthio, propylthio, isopropylthio, butylthio,    sec-butylthio, tert-butylthio, pentylthio, hexylthio),-   (20) 5- to 14-membered aromatic heterocyclic groups (including,    e.g., 5- to 6-membered monocyclic aromatic heterocyclic groups such    as thienyl, furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl,    isothiazolyl, oxazolyl, isooxazolyl, pyridyl, pyrazinyl,    pyrimidinyl, pyridazinyl, 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl,    1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, triazolyl, tetrazolyl, and    triazinyl; and-   8- to 14-membered condensed polycyclic (preferably, bicyclic or    tricyclic) aromatic heterocyclic groups such as benzothiophenyl,    benzofuranyl, benzoimidazolyl, benzooxazolyl, benzoisooxazolyl,    benzothiazolyl, benzoisothiazolyl, benzotriazolyl, imidazopyridinyl,    thienopyridinyl, furopyridinyl, pyrrolopyridinyl, pyrazolopyridinyl,    oxazolopyridinyl, thiazolopyridinyl, imidazopyrazinyl,    imidazopyrimidinyl, thienopyrimidinyl, furopyrimidinyl,    pyrrolopyrimidinyl, pyrazolopyrimidinyl, oxazolopyrimidinyl,    thiazolopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thienyl,    phenoxathiinyl, indolyl, isoindolyl, 1H-indazolyl, purinyl,    isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl,    quinazolinyl, cinnolinyl, carbazolyl, β-carbolinyl, phenanthridinyl,    acridinyl, phenazinyl, phenothiazinyl, and phenoxazinyl),-   (21) 3- to 14-membered non-aromatic heterocyclic groups (including,    e.g., 3- to 8-membered monocyclic non-aromatic heterocyclic groups    such as aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl,    thietanyl, tetrahydrothienyl, tetrahydrofuranyl, pyrrolinyl,    pyrrolidinyl, imidazolinyl, imidazolidinyl, oxazolinyl,    oxazolidinyl, pyrazolinyl, pyrazolidinyl, thiazolinyl,    thiazolidinyl, tetrahydroisothiazolyl, tetrahydrooxazolyl,    tetrahydroisooxazolyl, piperidinyl, piperazinyl,    tetrahydropyridinyl, dihydropyridinyl, dihydrothiopyranyl,    tetrahydropyrimidinyl, tetrahydropyridazinyl, dihydropyranyl,    tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl,    thiomorpholinyl, azepanyl, diazepanyl, azepinyl, oxepanyl, azocanyl,    and diazocanyl; and 9- to 14-membered condensed polycyclic    (preferably, bicyclic or tricyclic) non-aromatic heterocyclic groups    such as dihydrobenzofuranyl, dihydrobenzoimidazolyl,    dihydrobenzooxazolyl, dihydrobenzothiazolyl,    dihydrobenzoisothiazolyl, dihydronaphtho[2,3-b]thienyl,    tetrahydroisoquinolyl, tetrahydroquinolyl, 4H-quinolizinyl,    indolinyl, isoindolinyl, tetrahydrothieno[2,3-c]pyridinyl,    tetrahydrobenzoazepinyl, tetrahydroquinoxalinyl,    tetrahydrophenanthridinyl, hexahydrophenothiazinyl,    hexahydrophenoxazinyl, tetrahydrophthalazinyl,    tetrahydronaphthyridinyl, tetrahydroquinazolinyl,    tetrahydrocinnolinyl, tetrahydrocarbazolyl, tetrahydro-β-carbolinyl,    tetrahydroacridinyl, tetrahydrophenazinyl, tetrahydrothioxanthenyl,    and octahydroisoquinolyl),-   (22) C₁₋₆ alkyl-carbonyl groups that may be halogenated (e.g.,    acetyl, chloroacetyl, trifluoroacetyl, trichloroacetyl, propanoyl,    butanoyl, pentanoyl, hexanoyl),-   (23) C₆₋₁₄ aryl-carbonyl groups (e.g., benzoyl, 1-naphthoyl,    2-naphthoyl),-   (24) 5- to 14-membered aromatic heterocyclic carbonyl groups (e.g.,    nicotinoyl, isonicotinoyl, thenoyl, furoyl),-   (25) 3- to 14-membered non-aromatic heterocyclic carbonyl groups    (e.g., morpholinylcarbonyl, piperidinylcarbonyl,    pyrrolidinylcarbonyl),-   (26) C₁₋₆ alkoxy-carbonyl groups (e.g., methoxycarbonyl,    ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl,    isobutoxycarbonyl, sec-butoxycarbonyl, tert-butoxycarbonyl,    pentyloxycarbonyl, hexyloxycarbonyl),-   (27) C₆₋₁₄ aryloxy-carbonyl groups (e.g., phenyloxycarbonyl,    1-naphthyloxycarbonyl, 2-naphthyloxycarbonyl),-   (28) C₇₋₁₆ aralkyloxy-carbonyl groups (e.g., benzyloxycarbonyl,    phenethyloxycarbonyl),-   (29) carboxy groups,-   (30) carbamoyl groups,-   (31) mono- or di-C₁₋₆ alkyl-carbamoyl groups (e.g., methylcarbamoyl,    ethylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl,    N-ethyl-N-methylcarbamoyl),-   (32) C₆₋₁₄ aryl-carbamoyl groups (e.g., phenylcarbamoyl),-   (33) 5- to 14-membered aromatic heterocyclic carbamoyl groups (e.g.,    pyridylcarbamoyl, thienylcarbamoyl),-   (34) 3- to 14-membered non-aromatic heterocyclic carbamoyl groups    (e.g., morpholinylcarbamoyl, piperidinylcarbamoyl),-   (35) C₁₋₆ alkylsulfonyl groups that may be halogenated    (methylsulfonyl, difluoromethylsulfonyl, trifluoromethylsulfonyl,    ethylsulfonyl, propylsulfonyl, isopropylsulfonyl, butylsulfonyl,    4,4,4-trifluorobutylsulfonyl, pentylsulfonyl, hexylsulfonyl),-   (36) C₆₋₁₄ arylsulfonyl groups (e.g., phenylsulfonyl,    1-naphthylsulfonyl, 2-naphthylsulfonyl),-   (37) 5- to 14-membered aromatic heterocyclic sulfonyl groups (e.g.,    pyridylsulfonyl, thienylsulfonyl),-   (38) C₁₋₆ alkylsulfinyl groups that may be halogenated (e.g.,    methylsulfinyl, ethylsulfinyl),-   (39) C₆₋₁₄ arylsulfinyl groups (e.g., phenylsulfinyl,    1-naphthylsulfinyl, 2-naphthylsulfinyl),-   (40) 5- to 14-membered aromatic heterocyclic sulfinyl groups (e.g.,    pyridylsulfinyl, thienylsulfinyl),-   (41) mono- or di-C₁₋₆ alkylamino groups (e.g., methylamino,    ethylamino, propylamino, isopropylamino, butylamino, dimethylamino,    diethylamino, dipropylamino, dibutylamino, N-ethyl-N-methylamino),-   (42) mono- or di-C₆₋₁₄ arylamino groups (e.g., phenylamino),-   (43) 5- to 14-membered aromatic heterocyclic amino groups (e.g.,    pyridylamino),-   (44) C₇₋₁₆ aralkylamino groups (e.g., benzylamino),-   (45) formylamino groups,-   (46) C₁₋₆ alkyl-carbonylamino groups (e.g., acetylamino,    propanoylamino, butanoylamino),-   (47) (C₁₋₆ alkyl)(C₁₋₆ alkyl-carbonyl) amino groups (e.g.,    N-acetyl-N-methylamino),-   (48) C₆₋₁₄ aryl-carbonylamino groups (e.g., phenylcarbonylamino,    naphthylcarbonylamino),-   (49) C₁₋₆ alkoxy-carbonylamino groups (e.g., methoxycarbonylamino,    ethoxycarbonylamino, propoxycarbonylamino, butoxycarbonylamino,    tert-butoxycarbonylamino),-   (50) C₇₋₁₆ aralkyloxy-carbonylamino groups (e.g.,    benzyloxycarbonylamino),-   (51) C₁₋₆ alkylsulfonylamino groups (e.g., methylsulfonylamino,    ethylsulfonylamino),-   (52) C₆₋₁₄ arylsulfonylamino groups that may be substituted with    C₁₋₆ alkyl groups (e.g., phenylsulfonylamino, toluenesulfonylamino),-   (53) amino groups,-   (54) C₂₋₆ alkenyl groups (e.g., ethenyl, 1-propenyl, 2-propenyl,    2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl,    3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl,    4-methyl-3-pentenyl, 1-hexenyl, 3-hexenyl, 5-hexenyl),-   (55) C₂₋₆ alkynyl groups (e.g., ethynyl, 1-propynyl, 2-propynyl,    1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl,    4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl,    4-methyl-2-pentynyl),-   (56) C₃₋₁₀ cycloalkyl group (e.g., cyclopropyl, cyclobutyl,    cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,    bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.2.1]octyl,    adamantyl),-   (57) C₃₋₁₀ cycloalkenyl groups (e.g., cyclopropenyl, cyclobutenyl,    cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl),-   (58) C₁₋₆ cycloalkoxy-carbonyl groups (e.g., cyclopentoxy)-   (59) C₇₋₁₆ aralkylthio groups (e.g., S-benzylthio)-   (60) mercapto groups,-   (61) sulfo groups, and-   (62) guanidino groups.

The number of substituents at substitutable positions of the “C₇₋₁₆aralkyl group that may have a substituent” represented by the A- is, forexample, 1 to 5, preferably 1 to 3. If the number of substituents is notless than 2, the substituents may be the same or different.

The substituents may be selected from the Substituent Group a.

The number of substituents at substitutable positions of the “aromaticheterocycle group that may have a substituent” represented by the A- is,for example, 1 to 5, preferably 1 to 3. If the number of substituents isnot less than 2, the substituents may be the same or different.

The substituents may be selected from C₁₋₆ alkyl groups that may behalogenated (e.g., methyl, chloromethyl, difluoromethyl,trichloromethyl, trifluoromethyl, ethyl, 2-bromoethyl,2,2,2-trifluoroethyl, tetrafluoroethyl, pentafluoroethyl, propyl,2,2-difluoropropyl, 3,3,3-trifluoropropyl, isopropyl, butyl,4,4,4-trifluorobutyl, isobutyl, sec-butyl, tert-butyl, pentyl,isopentyl, neopentyl, 5,5,5-trifluoropentyl, hexyl,6,6,6-trifluorohexyl), C₆₋₁₄ aryl groups (e.g., phenyl, 1-naphthyl,2-naphthyl, 1-anthryl, 2-anthryl, 9-anthryl), C₇₋₁₆ aralkyl groups(e.g., benzyl, phenethyl, naphthylmethyl, phenylpropyl), and theSubstituent Group a.

Examples of the “derivative having a C₃₋₈ carbon chain” represented bythe A- include, for example, C₃₋₈ alkyl groups that may have asubstituent (e.g., propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, isopentyl, neopentyl, 1-ethylpropyl, hexyl,isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl,2-ethylbutyl, heptyl, octyl), C₃₋₁₀ cycloalkyl groups (e.g.,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cyclooctyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl,bicyclo[3.2.1]octyl, adamantyl), and the like.

Examples of the alkyl groups “C₃₋₈ alkyl groups” of the “C₃₋₈ alkylgroups that may have a substituent” represented by the A- are preferablypropyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, andisopentyl.

The number of substituents at substitutable positions of the “C₃₋₈ alkylgroup that may have a substituent” represented by the A- is, forexample, 1 to 5, preferably 1 to 3. If the number of substituents is notless than 2, the substituents may be the same or different.

The substituents may be selected from C₆₋₁₄ aryl groups (e.g., phenyl,1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl, 9-anthryl), and theSubstituent Group a.

Examples of the substituent of the “C₃₋₈ alkyl groups that may have asubstituent” represented by the A- are, preferably hydroxy groups,mercapto groups, amino groups, C₆₋₁₄ aryl groups, C₇₋₁₆ aralkylthiogroups, guanidino groups, C₁₋₆ alkylthio groups that may be halogenated,C₁₋₆ alkoxy-carbonyl groups, C₁₋₆ cycloalkoxy-carbonyl groups, C₇₋₁₆aralkyloxy-carbonyl groups, carboxy groups, and carbamoyl groups.

Examples of the “derivative having a C₈₋₂₂ linear fatty chain”represented by the A- include, for example, linear C₈₋₂₂ alkyl groups(e.g., octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl,pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl,heneicosylidene, docosyl) and the like.

R¹ of the “derivative represented by R¹—(CHR²)—” may be selected from ahydrogen atom or may be selected from the following Substituent Group b.

[Substituent Group b]

-   (1) halogen atoms,-   (2) cyano groups,-   (3) hydroxy groups,-   (4) C₁₋₆ alkoxy groups that may have a substituent (e.g., methoxy,    ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy,    tert-butoxy, pentyloxy, hexyloxy),-   (5) C₆₋₁₄ aryloxy groups that may have a substituent (e.g., phenoxy,    naphthoxy),-   (6) C₇₋₁₆ aralkyloxy groups that may have a substituent (e.g.,    benzyloxy),-   (7) C₁₋₆ alkyl-carbonyloxy groups that may have a substituent (e.g.,    acetoxy, propanoyloxy),-   (8) C₁₋₆ alkyl-carbonyl groups that may have a substituent (e.g.,    acetyl, chloroacetyl, trifluoroacetyl, trichloroacetyl, propanoyl,    butanoyl, pentanoyl, hexanoyl),-   (9) C₆₋₁₄ aryl-carbonyl groups that may have a substituent (e.g.,    benzoyl, 1-naphthoyl, 2-naphthoyl),-   (10) 5- to 14-membered aromatic heterocyclic carbonyl groups that    may have a substituent (e.g., nicotinoyl, isonicotinoyl, thenoyl,    furoyl),-   (11) 3- to 14-membered non-aromatic heterocyclic carbonyl groups    that may have a substituent (e.g., morpholinylcarbonyl,    piperidinylcarbonyl, pyrrolidinylcarbonyl),-   (12) C₁₋₆ alkoxy-carbonyl groups that may have a substituent (e.g.,    methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl,    isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl,    sec-butoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl,    hexyloxycarbonyl),-   (13) C₆₋₁₄ aryloxy-carbonyl groups that may have a substituent    (e.g., phenyloxycarbonyl, 1-naphthyloxycarbonyl,    2-naphthyloxycarbonyl),-   (14) C₇₋₁₆ aralkyloxy-carbonyl groups that may have a substituent    (e.g., benzyloxycarbonyl, phenethyloxycarbonyl),-   (15) carboxy groups,-   (16) carbamoyl groups,-   (17) mono- or di-C₁₋₆ alkyl-carbamoyl groups that may have a    substituent (e.g., methylcarbamoyl, ethylcarbamoyl,    dimethylcarbamoyl, diethylcarbamoyl, N-ethyl-N-methylcarbamoyl),-   (18) C₆₋₁₄ aryl-carbamoyl groups that may have a substituent (e.g.,    phenylcarbamoyl),-   (19) C₁₋₆ alkylthio groups that may have a substituent (e.g.,    methylthio, ethylthio, propylthio, isopropylthio, butylthio,    sec-butylthio, tert-butylthio, pentylthio, hexylthio),-   (20) C₁₋₆ alkylsulfonyl groups that may have a substituent    (methylsulfonyl, ethylsulfonyl, propylsulfonyl, isopropylsulfonyl,    butylsulfonyl, pentylsulfonyl, hexylsulfonyl),-   (21) C₁₋₆ alkylsulfinyl groups that may have a substituent (e.g.,    methylsulfinyl, ethylsulfinyl),-   (22) mono- or di-C₁₋₆ alkylamino groups that may have a substituent    (e.g., methylamino, ethylamino, propylamino, isopropylamino,    butylamino, dimethylamino, diethylamino, dipropylamino,    dibutylamino, N-ethyl-N-methylamino),-   (23) mono- or di-C₆₋₁₄ arylamino groups that may have a substituent    (e.g., phenylamino),-   (24) C₁₋₆ alkyl-carbonylamino groups that may have a substituent    (e.g., acetylamino, propanoylamino, butanoylamino),-   (25) C₆₋₁₄ aryl-carbonylamino groups that may have a substituent    (e.g., phenylcarbonylamino, naphthylcarbonylamino),-   (26) C₁₋₆ alkoxy-carbonylamino groups that may have a substituent    (e.g., methoxycarbonylamino, ethoxycarbonylamino,    propoxycarbonylamino, butoxycarbonylamino,    tert-butoxycarbonylamino),-   (27) C₇₋₁₆ aralkyloxy-carbonylamino groups that may have a    substituent (e.g., benzyloxycarbonylamino),-   (28) 5 to 14-membered aromatic heterocyclic groups that may have a    substituent (including, e.g., 5- to 6-membered monocyclic aromatic    heterocyclic groups such as thienyl, furyl, pyrrolyl, imidazolyl,    pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isooxazolyl, pyridyl,    pyrazinyl, pyrimidinyl, pyridazinyl, 1,2,4-oxadiazolyl,    1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl,    triazolyl, tetrazolyl, and triazinyl; and-   8- to 14-membered condensed polycyclic (preferably, bicyclic or    tricyclic) aromatic heterocyclic groups such as benzothiophenyl,    benzofuranyl, benzoimidazolyl, benzooxazolyl, benzoisooxazolyl,    benzothiazolyl, benzoisothiazolyl, benzotriazolyl, imidazopyridinyl,    thienopyridinyl, furopyridinyl, pyrrolopyridinyl, pyrazolopyridinyl,    oxazolopyridinyl, thiazolopyridinyl, imidazopyrazinyl,    imidazopyrimidinyl, thienopyrimidinyl, furopyrimidinyl,    pyrrolopyrimidinyl, pyrazolopyrimidinyl, oxazolopyrimidinyl,    thiazolopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thienyl,    phenoxathiinyl, indolyl, isoindolyl, 1H-indazolyl, purinyl,    isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl,    quinazolinyl, cinnolinyl, carbazolyl, β-carbolinyl, phenanthridinyl,    acridinyl, phenazinyl, phenothiazinyl, and phenoxazinyl),-   (29) C₆₋₁₄ aryl groups that may have a substituent (e.g., phenyl,    1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl, 9-anthryl),-   (30) C₇₋₁₆ aralkylthio groups (e.g., S-benzylthio),-   (31) sulfo groups, and-   (32) mercapto groups.

A substituent may be selected from the Substituent Group a for each ofthe respective substituents of the C₁₋₆ alkoxy groups that may have asubstituent, the C₆₋₁₄ aryloxy groups that may have a substituent, theC₇₋₁₆ aralkyloxy groups that may have a substituent, the C₁₋₆alkyl-carbonyloxy groups that may have a substituent, the C₁₋₆alkyl-carbonyl groups that may have a substituent, the C₆₋₁₄aryl-carbonyl groups that may have a substituent, the 5- to 14-memberedaromatic heterocyclic carbonyl groups that may have a substituent, the3- to 14-membered non-aromatic heterocyclic carbonyl groups that mayhave a substituent, the C₁₋₆ alkoxy-carbonyl groups that may have asubstituent, the C₆₋₁₄ aryloxy-carbonyl groups that may have asubstituent, the C₇₋₁₆ aralkyloxy-carbonyl groups that may have asubstituent, the mono- or di-C₁₋₆ alkyl-carbamoyl groups that may have asubstituent, the C₆₋₁₄ aryl-carbamoyl groups that may have asubstituent, the C₁₋₆ alkylthio groups that may have a substituent, theC₁₋₆ alkylsulfonyl groups that may have a substituent, the C₁₋₆alkylsulfinyl groups that may have a substituent, the mono- or di-C₁₋₆alkylamino groups that may have a substituent, the mono- or di-C₆₋₁₄arylamino groups that may have a substituent, the C₁₋₆alkyl-carbonylamino groups that may have a substituent, the C₆₋₁₄aryl-carbonylamino groups that may have a substituent, the C₁₋₆alkoxy-carbonylamino groups that may have a substituent, the C₇₋₁₆aralkyloxy-carbonylamino groups that may have a substituent, the 5 to14-membered aromatic heterocyclic groups that may have a substituent,and C₆₋₁₄ aryl groups that may have a substituent in the [SubstituentGroup b].

R¹ of the “derivative represented by R¹—(CHR²)—” is preferably ahydrogen atom and a C₆₋₁₄ aryl group that may have a substituent, morepreferably phenyl.

R² of the “derivative represented by R¹—(CHR²)—” may be selected fromthe following Substituent Group c.

[Substituent Group c]

-   (1) C₁₋₆ alkoxy-carbonyl groups (e.g., methoxycarbonyl,    ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl,    isobutoxycarbonyl, sec-butoxycarbonyl, tert-butoxycarbonyl,    pentyloxycarbonyl, hexyloxycarbonyl),-   (2) C₂₋₆ cycloalkoxy-carbonyl groups (e.g., cyclopentoxy)-   (3) C₆₋₁₄ aryloxy-carbonyl groups (e.g., phenyloxycarbonyl,    1-naphthyloxycarbonyl, 2-naphthyloxycarbonyl),-   (4) C₇₋₁₆ aralkyloxy-carbonyl groups (e.g., benzyloxycarbonyl,    phenethyloxycarbonyl),-   (5) carboxy groups,-   (6) carbamoyl groups,-   (7) mono- or di-C₁₋₆ alkyl-carbamoyl groups (e.g., methylcarbamoyl,    ethylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl,    N-ethyl-N-methylcarbamoyl), and-   (8) C₆₋₁₄ aryl-carbamoyl groups (e.g., phenylcarbamoyl).

R² of the “derivative represented by R¹—(CHR²)—” is preferably a C₁₋₆alkoxy-carbonyl group, a C₁₋₆ cycloalkoxy-carbonyl group, a C₇₋₁₆aralkyloxy-carbonyl group, a carboxy group, a carbamoyl group, morepreferably a C₁₋₆ alkoxy-carbonyl group, and most preferablyethoxycarbonyl.

R³ of the “derivative represented by R³—(CR⁴R⁵)—(CR⁶R⁷)—” may beselected from a hydrogen atom or the Substituent Group b.

R³ of the “derivative represented by R³—(CR⁴R⁵)—(CR⁶R⁷)—” is preferablya hydrogen atom, a hydroxy group, a mercapto group, an amino group, a 5-to 14-membered aromatic heterocyclic group that may have a substituent,a C₇₋₁₆ aralkylthio group, and a C₆₋₁₄ aryl group that may have asubstituent, more preferably, a hydrogen atom, a hydroxy group, amercapto group, an amino group, imidazolyl that may have a substituent,indolyl that may have a substituent, S-benzylthio, and phenyl that mayhave a substituent, particularly preferably indolyl, S-benzylthio, andphenyl that may have a substituent. Phenyl is most preferable.

R⁴ and R⁵ of the “derivative represented by R³—(CR⁴R⁵)—(CR⁶R⁷)—” mayindependently be selected from a hydrogen atom or the Substituent Groupa.

R⁴ and R⁵ of the “derivative represented by R³—(CR⁴R⁵)—(CR⁶R⁷)—” arepreferably hydrogen atoms.

R⁶ of the “derivative represented by R³—(CR⁴R⁵)—(CR⁶R⁷)—” may beselected from a hydrogen atom or the following Substituent Group d.

[Substituent Group d]

-   (1) hydroxy groups,-   (2) C₁₋₆ alkyl groups that may be halogenated (e.g., methyl,    chloromethyl, difluoromethyl, trichloromethyl, trifluoromethyl,    ethyl, 2-bromoethyl, 2,2,2-trifluoroethyl, tetrafluoroethyl,    pentafluoroethyl, propyl, 2,2-difluoropropyl, 3,3,3-trifluoropropyl,    isopropyl, butyl, 4,4,4-trifluorobutyl, isobutyl, sec-butyl,    tert-butyl, pentyl, isopentyl, neopentyl, 5,5,5-trifluoropentyl,    hexyl, 6,6,6-trifluorohexyl),-   (3) C₃₋₁₀ cycloalkyl groups (e.g., cyclopropyl, cyclobutyl,    cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,    bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl, bicyclo[3.2.1]octyl,    adamantyl),-   (4) 5- to 14-membered aromatic heterocyclic groups that may have a    substituent (including, e.g., 5- to 6-membered monocyclic aromatic    heterocyclic groups such as thienyl, furyl, pyrrolyl, imidazolyl,    pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, isooxazolyl, pyridyl,    pyrazinyl, pyrimidinyl, pyridazinyl, 1,2,4-oxadiazolyl,    1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl,    triazolyl, tetrazolyl, and triazinyl; and-   8- to 14-membered condensed polycyclic (preferably, bicyclic or    tricyclic) aromatic heterocyclic groups such as benzothiophenyl,    benzofuranyl, benzoimidazolyl, benzooxazolyl, benzoisooxazolyl,    benzothiazolyl, benzoisothiazolyl, benzotriazolyl, imidazopyridinyl,    thienopyridinyl, furopyridinyl, pyrrolopyridinyl, pyrazolopyridinyl,    oxazolopyridinyl, thiazolopyridinyl, imidazopyrazinyl,    imidazopyrimidinyl, thienopyrimidinyl, furopyrimidinyl,    pyrrolopyrimidinyl, pyrazolopyrimidinyl, oxazolopyrimidinyl,    thiazolopyrimidinyl, pyrazolotriazinyl, naphtho[2,3-b]thienyl,    phenoxathiinyl, indolyl, isoindolyl, 1H-indazolyl, purinyl,    isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl,    quinazolinyl, cinnolinyl, carbazolyl, β-carbolinyl, phenanthridinyl,    acridinyl, phenazinyl, phenothiazinyl, and phenoxazinyl), and-   (5) C₆₋₁₄ aryl groups that may have a substituent (e.g., phenyl,    1-naphthyl, 2-naphthyl, 1-anthryl, 2-anthryl, 9-anthryl).

A substituent may be selected from the Substituent Group a for each ofthe respective substituents of the 5- to 14-membered aromaticheterocyclic groups that may have a substituent, and the C₆₋₁₄ arylgroups that may have a substituent in the [Substituent Group d].

R⁶ of the “derivative represented by R³—(CR⁴R⁵)—(CR⁶R⁷)—” is preferablya hydrogen atom and methyl, and most preferably a hydrogen atom.

R⁷ of the “derivative represented by R³—(CR⁴R⁵)—(CR⁶R⁷)—” may beselected from the Substituent Group c.

R⁷ of the “derivative represented by R³—(CR⁴R⁵)—(CR⁶R⁷)—” is preferablya C₁₋₆ alkoxy-carbonyl group, a C₁₋₆ cycloalkoxy-carbonyl group, a C₇₋₁₆aralkyloxy-carbonyl group, a carboxy group, a carbamoyl group, morepreferably a C₁₋₆ alkoxy-carbonyl group, most preferably ethoxycarbonyl.

The “A-” is preferably the derivative having a carbon chain, thederivative represented by R¹—(CHR²)—, and the derivative represented byR³—(CR⁴R⁵)—(CR⁶R⁷)—, and more preferably, the derivative represented byR³—(CR⁴R⁵)—(CR⁶R⁷)—.

A hydrophobic primary amine compound represented by Formula (I): A-NH₂[wherein A denotes a hydrophobic moiety] is preferably an α-amino acidderivative that may have a substituent. The amino acid may be theL-isomer, the D-isomer, or the mixture thereof.

Examples of the “α-amino acid” of the “α-amino acid that may have asubstituent” used in the present invention include, for example,alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, 2-aminomalonic acid, 2-aminoadipic acid, glycine,histidine, isoleucine, leucine, lysine, ornithine, 2,4-diaminobutyricacid, methionine, phenylalanine, serine, threonine, tryptophan,5-methyltryptophan, tyrosine, valine, alloisoleucine, norvaline,norleucine, tert-leucine, γ-methylleucine, phenylglycine, 2-aminobutyricacid, cysteic acid, homocysteic acid, 1-naphthylalanine,2-naphthylalanine, 2-thienylglycine, 3-thienylglycine,3-benzothienylalanine, 4-biphenylalanine, pentamethylphenylalanine,1-aminocyclopropane-1-carboxylic acid, 1-aminocyclobutane-1-carboxylicacid, 1-aminocyclopentane-1-carboxylic acid,1-aminocyclohexane-1-carboxylic acid, 1-aminocycloheptane-1-carboxylicacid, and the like. The “α-amino acid” is preferably phenylalanine,phenylglycine, isoleucine, leucine, tyrosine, tryptophan, cysteine,serine, and threonine. Phenylalanine is particularly preferable.

The “α-amino acid” used in the present invention may have 1 to 3substituents at substitutable positions. Examples of the substituentsinclude halogen atoms, nitro groups, C₁₋₆ alkyl groups that may besubstituted with 1 to 3 halogen atoms, C₇₋₁₃ aralkyl groups that may besubstituted with 1 to 3 halogen atoms (e.g., benzyl), C₁₋₆ alkoxygroups, and the like.

In this description, a “derivative” means a compound and acharacteristic group generated by changing a small part in a molecule ofa certain compound by introduction of a function group, oxidation,reduction, substitution of atoms, and specific examples include, forexample, carboxylic acid, C₁₋₁₈ alkyl ester, C₆₋₁₈ aromatic ester, C₃₋₁₈cycloalkyl ester, C₁₋₁₈ alkyl monosubstituted amide, C₁₋₁₈ alkyldisubstituted amide, unsubstituted amide, and the like of a certaincompound. Examples of the C₁₋₁₈ alkyl substituents include methyl,ethyl, n-propyl, i-propyl, cyclopropyl, n-butyl, sec-butyl, t-butyl,cyclopropylmethyl, cyclobutyl, n-pentyl, 1-methylbutyl, 2-methylbutyl,3-methylbutyl, 2,2-dimethylpropyl, 2,3-dimethylpropyl,3,3-dimethylpropyl, cyclopropylethyl, cyclobutylmethyl, cyclopropyl,n-hexyl, n-heptyl, n-octyl, n-nonanyl, n-decanyl, n-hexadecanyl,n-octadecanyl, and the like.

The “derivative” may include substitution with halogen atoms such asfluorine, chlorine, bromine, and iodine, or nitro groups and the like.

The “derivative” used in the present invention is preferably C₁₋₁₈ alkylester.

The α-amino acid derivative is preferably a phenylalanine derivative andis preferably phenylalanine alkyl ester, particularly, phenylalanineethyl ester. The amino acid derivative may be the L-isomers, theD-isomers, or the mixture thereof.

Examples of the salt of the hydrophobic primary amine compoundrepresented by Formula (I) include the same salts as those illustratedas the salt of the poly(amino acid).

In this description, the “graft copolymer” refers to a graft copolymerrepresented by Formula (VI):

[wherein M is alkali metal, A is a hydrophobic moiety, and n is aninteger from 10 to 100,000, diagonal lines intervening three monomerunits represent that the monomer units are arranged in irregular order;and x is a mole fraction of the monomer unit represented by Formula(III):

y is a mole fraction of the monomer unit represented by Formula (IV):

z is a mole fraction of the monomer unit represented by Formula (V):

x, y, and z satisfy the following equations:0≤x<1;0≤y<1;0≤z<1; andx+y+z=1];  [Math. 2]

An amount of the hydrophobic primary amine compound or a salt thereofrepresented by Formula (I) used at step (1) is typically 0.01 to 5equivalents, preferably 0.1 to 1.5 equivalents, relative to thepoly(amino acid).

Examples of a condensing agent used at step (1) include a condensingagent used in usual peptide synthesis and include, for example,water-soluble carbodiimide hydrochloride [e.g.,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride] (WSC.HCl),4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloriden-hydrate (DMT-MM),N,N,N′,N′-tetramethyl-O—(N-succinimidyl)uroniumtetrafluoroborate (TSTU),2-(5-norbornene-2,3-dicarboximide)-1,1,3,3-tetramethyluroniumtetrafluoroborate(TNTU), and the like.

An amount of the condensing agent used at step (1) is typically 0.01 to5 equivalents, preferably 0.1 to 1.5 equivalents, relative to thepoly(amino acid).

Step (1) is performed in a solvent not affecting a reaction. Examples ofsuch a solvent include, for example, water, a mixture of an organicsolvent described below and water, and the like. Examples of the organicsolvent include C₁₋₃ alcohol (e.g., methanol, ethanol, isopropanol,n-propanol), dimethylsulfoxide (DMSO), tetrahydrofuran (THF),N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc),N-methylpyrrolidone (NMP), aprotic solvents with high polarity such asacetonitrile, and the like.

If the poly(amino acid) is a free form, a base or a salt thereof isadded at step (1). Examples of the base used in this case include alkalimetal salts, carbonates, hydrogencarbonates, organic bases, and thelike.

If an alkali metal salt is used as the base at step (1), examplesthereof include sodium hydroxide, potassium hydroxide, lithiumhydroxide, and the like, and sodium hydroxide is preferable.

If a carbonate is used as the base at step (1), examples thereof includelithium carbonate, sodium carbonate, potassium carbonate, and the like,and sodium carbonate is preferable.

If a hydrogencarbonate is used as the base at step (1), examples thereofinclude, for example, lithium hydrogencarbonate, sodiumhydrogencarbonate, potassium hydrogencarbonate, and the like, and sodiumhydrogencarbonate is preferable.

If an organic base is used as the base at step (1), for example,trimethylamine, triethylamine, pyridine, picoline, triethanolamine,N-methylmorpholine, N,N-diisopropylethylamine,1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) may be used as needed.

An amount of the base or a salt thereof used at step (1) is typically0.01 to 100 equivalents, preferably 0.3 to 10 equivalents, morepreferably 0.5 to 1.5 equivalents, relative to the poly(amino acid).

If the poly(amino acid) is the salt described above or a sodium salt,the poly(amino acid) may directly be used for the reaction.

A reaction temperature of step (1) is typically −30 to 80° C.,preferably −5 to 45° C., more preferably −5 to 30° C.

A reaction time of step (1) is typically from 0.5 hours to 7 days,preferably from 1 hour to 2 days.

The graft copolymer acquired at step (1) may be subjected to knownpurification means for concentration, extraction, chromatography,ultrafiltration, centrifugal concentration, and the like or may directlybe used at the next step.

“Allowing an acid to act” at step (2) means that a deprotonated carboxylgroup (—COO⁻) contained in the graft copolymer is protonated into thestate of a carboxyl group (—COOH) by adding the acid.

Examples of the acid used at step (2) include inorganic acids such ashydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, andphosphoric acid; and organic acids such as formic acid, acetic acid,trifluoroacetic acid, fumaric acid, oxalic acid, tartaric acid, maleicacid, citric acid, succinic acid, malic acid, methanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid, lactic acid, and benzoicacid and, among them, hydrochloric acid, hydrobromic acid, and aceticacid are preferable.

Step (2) is typically performed in a solvent not affecting a reaction.Examples of such a solvent include, for example, water, a mixture of anorganic solvent described below and water, and the like. Examples of theorganic solvent include C₁₋₃ alcohol, dimethylsulfoxide (DMSO),tetrahydrofuran (THF), N,N-dimethylformamide (DMF),N,N-dimethylacetamide (DMAc), N-methylpyrrolidone (NMP), aproticsolvents with high polarity such as acetonitrile, and the like.Alternatively, step (2) may be performed without a solvent.

A reaction temperature of step (2) is typically −5 to 80° C., preferably−5 to 30° C.

In another preferable embodiment, a reaction temperature of step (2) is0 to 80° C.

This reaction may be conducted under an elevated temperature conditionor a lowered temperature condition as long as the temperature is withinthe temperature ranges.

The reaction temperature of this reaction is more preferably 40 to 70°C.

In this description, a “room temperature” is 1 to 30° C. unlessotherwise stated.

A reaction time of step (2) is typically from 0.5 hours to 7 days,preferably from 1 hour to 2 days.

In a preferred embodiment, the reaction is preferably conducted by usinghydrochloric acid at 60° C.

In the method, an introduction rate of the hydrophobic primary aminecompound represented by Formula (I) relative to the poly(amino acid) ispreferably 1 to 99%, more preferably 5 to 85%.

The poly(amino acid) is preferably poly(γ-glutamic acid).

The hydrophobic primary amine compound represented by Formula (I) ispreferably α-amino acid derivatives, particularly preferably,phenylalanine derivatives, and phenylalanine ethyl ester is especiallypreferable.

A number average molecular weight of the graft copolymer acquired inthis way is 1 to 2000 kDa, preferably 10 to 1000 kDa.

A second aspect of the present invention relates to an ionized graftcopolymer of a poly(amino acid) selected from the group consisting ofpoly(γ-glutamic acid), poly(α-glutamic acid), and poly(aspartic acid) ora salt thereof and a hydrophobic primary amine compound represented byFormula (I): A-NH₂ [wherein A denotes a hydrophobic moiety] or a saltthereof, and a production method thereof.

The production method of the ionized graft copolymer according to thesecond aspect of the present invention comprises the step of:

ionizing the graft copolymer by allowing a hydroxide of alkali metal, acarbonate of alkali metal, a hydrogencarbonate of alkali metal, aphosphate of alkali metal, a monohydrogen phosphate of alkali metal, adihydrogen phosphate of alkali metal, an organic acid salt of alkalimetal, or an acidic amino-acid salt of alkali metal to act on the graftcopolymer of the poly(amino acid) or a salt thereof and the hydrophobicprimary amine compound or a salt thereof.

The graft copolymer of a poly(amino acid) or a salt thereof and ahydrophobic primary amine compound or a salt thereof used as a rawmaterial at the step may be produced in conformity to the method ofproducing the graft copolymer of a poly(amino acid) or a salt thereofand a hydrophobic primary amine compound or a salt thereof describedabove or the method described in Patent Document 3.

The poly(amino acid) or a salt thereof is preferably poly(γ-glutamicacid).

The hydrophobic primary amine compound represented by Formula (I) ispreferably α-amino acid derivatives, particularly preferably,phenylalanine derivatives, and phenylalanine ethyl ester is especiallypreferable.

At this step, “allowing a hydroxide of alkali metal, a carbonate ofalkali metal, a hydrogencarbonate of alkali metal, a phosphate of alkalimetal, a monohydrogen phosphate of alkali metal, a dihydrogen phosphateof alkali metal, an organic acid salt of alkali metal, or an acidicamino-acid salt of alkali metal to act” means that a carboxyl group(—COOH) contained in the graft copolymer is deprotonated into the stateof a deprotonated carboxyl group (—COO).

Examples of the hydroxide of alkali metal used at this step include, forexample, lithium hydroxide, sodium hydroxide, potassium hydroxide, andthe like, and sodium hydroxide and potassium hydroxide are preferable.Potassium hydroxide is more preferable.

An amount of the hydroxide of alkali metal used at this step istypically 0.001 to 10 equivalents relative to the graft copolymer.

Examples of the carbonate of alkali metal used at this step include, forexample, lithium carbonate, sodium carbonate, potassium carbonate, andthe like, and sodium carbonate and potassium carbonate are preferable.Potassium carbonate is more preferable.

An amount of the carbonate of alkali metal used at this step istypically 0.001 to 10 equivalents relative to the graft copolymer.

Examples of the hydrogencarbonate of alkali metal used at this stepinclude, for example, lithium hydrogencarbonate, sodiumhydrogencarbonate, potassium hydrogencarbonate, and the like, and sodiumhydrogencarbonate and potassium hydrogencarbonate are preferable.Potassium hydrogencarbonate is more preferable.

An amount of the hydrogencarbonate of alkali metal used at this step is0.001 to 10 equivalents relative to the graft copolymer.

Examples of the phosphate of alkali metal used at this step include, forexample, lithium phosphate, sodium phosphate, potassium phosphate, andthe like, and sodium phosphate and potassium phosphate are preferable.Potassium phosphate is more preferable.

An amount of the phosphate of alkali metal used at this step istypically 0.001 to 10 equivalents relative to the graft copolymer.

Examples of the monohydrogen phosphate of alkali metal used at this stepinclude, for example, lithium monohydrogen phosphate, sodiummonohydrogen phosphate, potassium monohydrogen phosphate, and the like,and sodium monohydrogen phosphate and potassium monohydrogen phosphatepreferable. Potassium monohydrogen phosphate is more preferable.

An amount of the monohydrogen phosphate of alkali metal used at thisstep is typically 0.001 to 10 equivalents relative to the graftcopolymer.

Examples of the dihydrogen phosphate of alkali metal used at this stepinclude, for example, lithium dihydrogen phosphate, sodium dihydrogenphosphate, potassium dihydrogen phosphate, and the like, and sodiumdihydrogen phosphate and potassium dihydrogen phosphate are preferable.Potassium dihydrogen phosphate is more preferable.

An amount of the dihydrogen phosphate of alkali metal used at this stepis typically 0.001 to 10 equivalents relative to the graft copolymer.

Examples of “organic acid salt” used for the organic acid salt of alkalimetal used at this step include salts with formic acid, acetic acid,fumaric acid, oxalic acid, tartaric acid, maleic acid, lactic acid,citric acid, succinic acid, malic acid, benzoic acid, and the like.Among them, lactic acid, acetic acid, citric acid, and benzoic acid arepreferable, and lactic acid, acetic acid, and benzoic acid are morepreferable.

Examples of the organic acid salt of alkali metal used at this stepinclude, for example, lithium formate, lithium acetate, lithiumfumarate, lithium oxalate, lithium tartrate, lithium maleate, lithiumlactate, lithium citrate, lithium succinate, lithium malate, lithiumbenzoate, sodium formate, sodium acetate, sodium fumarate, sodiumoxalate, sodium tartrate, sodium maleate, sodium lactate, sodiumcitrate, sodium succinate, sodium malate, sodium benzoate, potassiumformate, potassium acetate, potassium fumarate, potassium oxalate,potassium tartrate, potassium maleate, potassium lactate, potassiumcitrate, potassium succinate, potassium malate, potassium benzoate, andthe like. Sodium lactate, sodium acetate, sodium benzoate, potassiumlactate, potassium acetate, and potassium benzoate are preferable and,among them, potassium lactate, potassium acetate, and potassium benzoateare preferable.

An amount of the organic acid salt of alkali metal used at this step istypically 0.001 to 10 equivalents relative to the graft copolymer.

Examples of “acidic amino acid” used for the acidic amino-acid salt ofalkali metal used at this step include aspartic acid, glutamic acid, andthe like. Among them, aspartic acid is preferable.

Examples of the acidic amino-acid salt of alkali metal used at this stepinclude, for example, lithium aspartate, lithium glutamate, sodiumaspartate, sodium glutamate, potassium aspartate, potassium glutamate,and the like. Sodium aspartate, and potassium aspartate are preferable.Potassium aspartate is more preferable.

An amount of the acidic amino-acid salt of alkali metal used at thisstep is typically 0.001″ to 10 equivalents relative to the graftcopolymer.

The step is performed in a solvent not affecting a reaction. Examples ofsuch a solvent include, for example, water, a mixture of an organicsolvent described below and water, and the like. Examples of the organicsolvent include C₁₋₃ alcohol, dimethylsulfoxide (DMSO), tetrahydrofuran(THF), N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAc),N-methylpyrrolidone (NMP), aprotic solvents with high polarity such asacetonitrile, acetone, pyridine, methyl acetate, and the like.

A reaction temperature of this step is typically −30 to 80° C.,preferably −5 to 45 more preferably −5 to 30° C.

A reaction time of this step is typically from 0.1 hours to 7 days,preferably from 0.5 hours to 2 days.

A number average molecular weight of the ionized graft copolymeracquired in this way is 1 to 2,000 kDa, preferably 10 to 1,000 kDa.

Examples of the ionized graft copolymer of the present inventionproduced by the method include an ionized graft copolymer represented byFormula (II):

[wherein M is alkali metal, A is a hydrophobic moiety, and n is aninteger from 10 to 100,000; diagonal lines intervening three monomerunits represent that the monomer units are arranged in irregular order;and x is a mole fraction of the monomer unit represented by Formula(III):

y is a mole fraction of the monomer unit represented by Formula (IV):

z is a mole fraction of the monomer unit represented by Formula (V):

andx, y, and z satisfy the following equations:0≤x<1;0<y<1;0<z<1; andx+y+z=1];  [Math. 3]

In the monomer unit represented by Formula (IV), examples of the alkalimetal denoted by M include lithium, sodium, potassium, and the like and,among them, the alkali metal is preferably sodium or potassium, mostpreferably potassium.

In the monomer unit represented by Formula (V), examples of thehydrophobic moiety denoted by A are the same as those of the“hydrophobic moiety” denoted by A in the hydrophobic primary aminecompound represented by Formula (I): A-NH₂ [wherein A denotes ahydrophobic moiety]. In the monomer unit represented by Formula (V),specific examples of —NH-A [wherein A denotes a hydrophobic moiety]include an α-amino acid derivative that may have a substituent. The“α-amino acid derivative” is preferably a phenylalanine derivative andis preferably a phenylalanine alkyl ester derivative, particularlypreferably a phenylalanine ethyl ester derivative. The “amino acidderivative” may be the L-isomer, the D-isomer, or the mixture thereof.

In Formula (II), the monomer unit represented by Formula (III)preferably has a mole fraction x of 0.01 to 0.99, more preferably 0.15to 0.95.

In Formula (II), the monomer unit represented by Formula (IV) preferablyhas a mole fraction y of 0.02 to 0.6, more preferably 0.05 to 0.5.

In Formula (II), the monomer unit represented Formula (V) preferably hasa mole fraction z of 0.01 to 0.99, more preferably 0.05 to 0.85.

In Formula (II), x, y, and z may have arbitrary numerical values withinthe respective preferable ranges, provided that x+y+z does not exceed 1.

The ionized copolymer is preferably balanced between moderate watersolubility and moderate hydrophobicity depending on the total number ofmonomer units in the graft copolymer and the structure of thehydrophobic moiety A for being used in production of nanoparticles thatare a third aspect of the present invention.

In this description, n denotes the total number of monomer units in thegraft copolymer.

In a preferred embodiment, n is 50 to 10,000, more preferably 100 to2,000.

In this description, a “hydrophobic parameter K” corresponds to anoctanol/water distribution coefficient when 1-octanol and water are usedas a solvent, and is indicated by Log Pow in this description.

A method of actually measuring Log Pow may be JIS-Z7260-107, forexample. However, since the ionized graft copolymer of poly(amino acid)of the present invention makes measurement difficult because of lowsolubility at the time of concentration measurement, a method may beemployed in which each of organic and water layers after distribution ishydrolyzed to quantitate the respective concentrations of amino acidmonomers.

The “hydrophobic parameter K” may be determined by using a calculationvalue from a CLOGP method, which is a method of calculation fromchemical structure, instead of an actual measurement value of Log Pow.The “hydrophobic parameter K” calculated with the CLOGP method isindicated by CLOGP in this description.

In a preferred embodiment, the hydrophobic parameter K is −15,000 to 0,more preferably −3,000 to 0.

A third aspect of the present invention relates to nanoparticlescontaining an ionized graft copolymer of a poly(amino acid) selectedfrom the group consisting of poly(γ-glutamic acid), poly(α-glutamicacid), and poly(aspartic acid) or a salt thereof and a hydrophobicprimary amine compound represented by Formula (I): A-NH₂ [wherein Adenotes a hydrophobic moiety] or a salt thereof, and a production methodthereof.

The production method of the nanoparticles according to the third aspectof the present invention comprises the steps of:

(1) condensing a poly(amino acid) selected from the group consisting ofpoly(γ-glutamic acid), poly(α-glutamic acid), and poly(aspartic acid) ora salt thereof and a hydrophobic primary amine compound represented byFormula (I): A-NH₂ [wherein A denotes a hydrophobic moiety] or a saltthereof;

(2) isolating a graft copolymer by allowing an acid to act on acondensate acquired at step (1);

(3) ionizing the graft copolymer by allowing a hydroxide of alkalimetal, a carbonate of alkali metal, or a hydrogencarbonate of alkalimetal to act on the graft copolymer isolated at step (2); and

(4) forming nanoparticles of the ionized graft copolymer acquired atstep (3).

Steps (1) and (2) may be performed in accordance with the productionmethod of a graft copolymer of a poly(amino acid) or a salt thereof anda hydrophobic primary amine compound represented by Formula (I) or asalt thereof described above.

Step (3) may be performed in accordance with the production method of anionized graft copolymer of a poly(amino acid) or a salt thereof and ahydrophobic primary amine compound represented by Formula (I) or a saltthereof described above.

The method of forming nanoparticles of the ionized graft copolymer ofstep (4) may be performed in conformity to the method described inPatent Document 3. For example, if a precipitation method is used, theionized graft copolymer acquired at step (3) may be dissolved in a goodsolvent and subsequently be mixed with a poor solvent to formnanoparticles.

The “good solvent” may be, for example, dimethyl sulfoxide or alcohols(such as methanol, ethanol, isopropanol, and n-propanol).

The “poor solvent” may be water. If water is used as the poor solvent,water is normally used as an aqueous solution of sodium chloride, sodiumphosphate, sodium monohydrogen phosphate, sodium dihydrogen phosphate,sodium carbonate, sodium hydrogencarbonate, potassium chloride,potassium phosphate, potassium monohydrogen phosphate, potassiumdihydrogen phosphate, potassium carbonate, potassium hydrogencarbonate,and the like. The solution of the ionized graft copolymer acquired atstep (3) and the poor solvent may be mixed in both batch-wise andcontinuous manners.

At the step of ionizing the graft copolymer of a poly(amino acid) or asalt thereof and a hydrophobic primary amine compound represented byFormula (I) or a salt thereof, the ionization may be controlled toadjust an ionization rate of COOH side chains of the poly(amino acid)not binding to the hydrophobic primary amine compound represented byFormula (I) or a salt thereof. By adjusting the ionization rate asdescribed above, nanoparticles with different properties may beproduced. Using the production method of the present inventionfacilitates scale-up and enables the large-scale synthesis of thenanoparticles. If the nanoparticles are prepared from the free form ofthe graft copolymer, an amount of ionization at the time of formation ofthe nanoparticles may be controlled. The type of ionization of the graftcopolymer is not limited to sodium and the ionization may selectively beachieved by various ion species.

In this description, the “free form of the graft copolymer” refers tothe case of y=0 in Formula (VI), i.e., the state in which all thecarboxyl groups in the graft copolymer are present as COOH.

In this description, the “ionized graft copolymer” refers to the case ofy is other than 0 in Formula (VI), i.e., the state in which all or someof the carboxyl groups in the graft copolymer are present as salt.

In this description, the “monomer unit” means a constituent unit ofpolymer such as a graft copolymer.

The “nanoparticle” means taking a form of particulates that are mainlycomposed of aggregates made of a graft copolymer of a poly(amino acid)or a salt thereof and a hydrophobic primary amine compound or a saltthereof and that have a size of 5000 nanometers (nm) or less in themajor axis while forming a clear interface with the surroundingenvironment.

The nanoparticles of the present invention may have various shapes suchas a spherical shape, a hollow shape, and a porous spherical shape.

A particle diameter of the nanoparticles of the present invention is 1nanometer (nm) to 1500 nm, preferably 1 nm to 500 nm, more preferably 10nm to 300 nm, under a physiological condition.

The “nanoparticles” of the present invention may contain a substanceother that the graft copolymer of a poly(amino acid) or a salt thereofand a hydrophobic primary amine compound or a salt thereof.

For example, the nanoparticles of the present invention may be used asan adjuvant for vaccine, when one or two or more antigens are containedin the nanoparticles or immobilized on the surfaces of thenanoparticles.

In this description, the “antigens” means those capable of inducing animmune reaction and may be, but not limited to, pathogens includingviruses such as human immunodeficiency virus (HIV) and humanpapillomavirus (HPV) and pathogenic organisms such as tubercule bacillusand tetanus bacillus or a portion thereof, or proteins, peptides, andnucleic acids, for example. Such antigens may be selected as neededdepending on a disease to be treated or prevented.

In this description, the “adjuvant” means a substance stimulating animmune system and enhancing an immune reaction.

Therefore, a forth aspect of the present invention relates to a vaccinecontaining such nanoparticles.

The vaccine of the present invention may contain the nanoparticlescontaining the graft copolymer of a poly(amino acid) and a hydrophobicprimary amine compound represented by Formula (I) as an adjuvant and theantigens, and may further contain a solvent, a solubilizing agent, asuspending agent, an isotonizing agent, a soothing agent, an antisepticagent, an anti-oxidizing agent, and the like.

Examples of the solvent include, for example, water for injection,alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil,and the like.

Examples of the solubilizing agent include, for example, polyethyleneglycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol,tris-aminomethane, cholesterol, triethanolamine, sodium carbonate,sodium citrate, and the like.

Examples of the suspending agent include, for example, surfactants suchas stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropioate, lecithin, benzalkonium chloride, benzethonium chloride, andglyceryl monostearate; hydrophilic macromolecules such as polyvinylalcohol, polyvinylpyrrolidone, carboxymethyl-cellulose sodium, methylcellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, andhydroxypropyl cellulose; magnesium chloride, and the like.

Examples of the isotonizing agent include, for example, glucose,D-sorbitol, sodium chloride, glycerin, D-mannitol, and the like.

Examples of the soothing agent include, for example, benzyl alcohol, andthe like.

Examples of the antiseptic agent include, for example, p-hydroxybenzoicacid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol,dehydroacetic acid, sorbic acid, and the like.

Examples of the anti-oxidizing agent include, for example, sulfite,ascorbic acid, α-tocopherol, and the like.

The vaccine of the present invention may be in any forms such assolution, suspension, lyophilizate, powder, capsules, and tablets. Ifthe vaccine of the present invention is solid, the vaccine may besuspended or dissolved in a proper solvent such as saline before use.

The vaccine of the present invention is biodegradable. Thebiodegradability means that a substance has structure degradable in vivoand that the substance itself and a degradation or metabolic productthereof are safe or are nontoxic or low-toxic.

With the vaccine of the present invention, mammals (e.g., mice, rats,rabbits, felines, canines, bovines, equines, monkeys, and humans) maysafely be inoculated.

An inoculum dose, an inoculation method, the number of times ofinoculation of the vaccine of the present invention may appropriately beselected depending on, for example, an age and a condition of a subject,a type of disease, a type of antigen, and the like.

The inoculum dose of the vaccine of the present invention is, forexample, an antigen amount of 1 mg to 100 mg per dose per adult (withbody weight of about 60 kg).

Examples of the inoculation method of the vaccine of the presentinvention include, for example, oral inoculation, subcutaneousinjection, intramuscular injection, infusion, and the like.

The number of times of inoculation of the vaccine of the presentinvention is from once to multiple times.

EXAMPLES

Although the present invention will hereinafter specifically bedescribed with examples, the present invention is not limited thereto. Agraft copolymer of poly(γ-glutamic acid) and phenylalanine ethyl esteris a polymer of the present invention and will hereinafter be referredto as γ-PGA-PAE. Water described in the following description may bereplaced with water for injection, ion-exchanged water, and the like.Unless otherwise stated, a molecular weight is a relative molecularweight and is a numerical value determined by a molecular weightmeasurement method using the following SEC-HPLC measurement: TSKgel α-M300×7.8 mm ID. (dual), 5 mM NaNO₃ DMSO:H₂O (9:1), 0.8 mL/minute, 40° C.,RI detector, standard: Pullulan (Shodex).

Example 1 Synthesis of γ-PGA-PAE

In a 1-L four-necked flask, distilled water (400 mL) and NaHCO₃ (8.4 g)were measured and dissolved at room temperature. To this solution, γ-PGA(12.1 g, 148 kDa) was added, washed and dissolved with distilled water(60 mL), and then ice-cooled. At ice temperature, WSC.HCl (19.2 g) wasadded and washed with distilled water (10 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (13.8 g) was added and washed with distilled water (15 mL).The solution was stirred for 1 hour at ice temperature and then stirredfor 24 hours at room temperature. At room temperature, 2 M hydrochloricacid (120 mL) was added dropwise and stirred for 2 hours. A precipitatewas sucked and filtered and was washed with distilled water (100 mL)twice. The precipitate was dried under reduced pressure at roomtemperature to acquire γ-PGA-PAE (23.2 g, yield: about 96.8%, PAEintroduction rate: 55%, moisture: 5.8%, 85 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.6-1.2 (brs, 3.6H), 1.4-2.4 (brm, 6.7H),2.6-2.8 (brm, 1.2H), 2.8-3.1 (brm, 3.0H), 3.8-4.5 (brm, 4.1H), 4.5-5.5(brm, 0.4H), 6.4-6.6 (brm, 0.1H), 6.7-7.5 (brm, 5.0H, a relative valuewhen the protons of the phenyl group of the phenylalanyl group areassumed as 5.0H), 7.6-8.7 (brm, 2.3H).

Example 2 Synthesis of γ-PGA-PAE

In a 1-L four-necked flask, distilled water (400 mL) and NaHCO₃ (8.4 g)were measured and dissolved at room temperature. To this solution, γ-PGA(12.1 g, 148 kDa) was added, washed and dissolved with distilled water(66 mL), and then ice-cooled. At ice temperature, WSC.HCl (19.2 g) wasadded and washed with distilled water (15 mL). After this solution wasstirred for 5 minutes, PAE.HCl (13.8 g) was added and washed withdistilled water (5 mL). The solution was stirred for 1 hour at icetemperature and then stirred for 5 hours at room temperature. At roomtemperature, 2 M hydrochloric acid (120 mL) was added dropwise andstirred for 2 hours. A precipitate was sucked and filtered and waswashed with distilled water (100 mL) twice. The precipitate was driedunder reduced pressure at room temperature to acquire γ-PGA-PAE (21.1 g,yield: about 94.5%, PAE introduction rate: 46%, moisture: 4.5%, 133kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.0H), 1.4-2.4 (brm, 6.2H),2.6-2.8 (brm, 0.8H), 2.8-3.1 (brm, 2.6H), 3.9-4.5 (brm, 4.0H), 4.5-5.5(brm, 0.4H), 6.4-6.6 (brm, 0.1H), 6.9-7.5 (brm, 5.0H, a relative valuewhen the protons of the phenyl group of the phenylalanyl group areassumed as 5.0H), 7.6-8.7 (brm, 2.2H).

Example 3 Synthesis of γ-PGA-PAE

In a 1-L four-necked flask, 1 M sodium hydroxide aqueous solution (90mL) was measured in distilled water (385 mL) at room temperature, andγ-PGA (12.1 g, 148 kDa) was dissolved and ice-cooled. At icetemperature, WSC.HCl (19.2 g) was added and washed with distilled water(4 mL). After this solution was stirred for 5 minutes, PAE.HCl (13.8 g)was added and washed with distilled water (6 mL). The solution wasstirred for 1 hour at ice temperature and then stirred for 5 hours atroom temperature. At room temperature, 2 M hydrochloric acid (120 mL)was added dropwise and stirred for 2 hours. A precipitate was sucked andfiltered and was washed with distilled water (100 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-PAE (21.7 g, yield: about 94.8%, PAE introduction rate:59%, moisture: 4.5%, 286 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 1.1-1.2 (brs, 3.2H), 1.4-2.4 (brm, 6.9H),2.6-2.8 (brm, 0.6H), 2.8-3.1 (brm, 2.4H), 3.1-3.8 (brm, 7.2H), 4.0 (brs,2.0H), 4.1-4.4 (brm, 2.5H), 4.6-5.2 (brm, 0.2H), 6.9-7.5 (brm, 5.0H, arelative value when the protons of the phenyl group of the phenylalanylgroup are assumed as 5.0H), 7.7-9.0 (brm, 2.6H), 10.0-15.0 (brs, 0.2H).

Example 4 Synthesis of γ-PGA-PAE

In a 1-L four-necked flask, 1 M sodium hydroxide aqueous solution (90mL) was measured in distilled water (385 mL) at room temperature, andγ-PGA (12.1 g, 148 kDa) was dissolved and ice-cooled. At icetemperature, WSC.HCl (19.2 g) was added and washed with distilled water(4 mL). After this solution was stirred for 5 minutes, PAE.HCl (13.8 g)was added and washed with distilled water (6 mL). The solution wasstirred for 1 hour at ice temperature and then stirred for 22 hours atroom temperature. At room temperature, 2 M hydrochloric acid (120 mL)was added dropwise and stirred for 2 hours. A precipitate was sucked andfiltered and was washed with distilled water (100 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-PAE (26.8 g, yield: about 93.6%, PAE introduction rate:59%, moisture: 23.6%, 44 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.7-1.2 (brs, 3.2H), 1.4-2.4 (brm, 6.8H),2.6-2.8 (brm, 1.1H), 2.8-3.1 (brm, 2.9H), 3.8-5.1 (brm, 4.4H), 6.9-7.5(brm, 5.0H, a relative value when the protons of the phenyl group of thephenylalanyl group are assumed as 5.0H), 7.6-8.7 (brm, 2.4H).

Example 5 Synthesis of γ-PGA-PAE

In a 1-L four-necked flask, distilled water (350 mL) and 1 M NaOH (90mL) were measured and mixed at room temperature. To this solution, γ-PGA[12.1 g, 65 kDa, D:L ratio (35:65)] was added and washed with distilledwater (28 mL). After dissolution, the solution was ice-cooled. At icetemperature, WSC.HCl (19.2 g) was added and washed with distilled water(10 mL). After this solution was stirred for 5 minutes, PAE.HCl (13.8 g)was added and washed with distilled water (10 mL). The solution wasstirred for 1 hour at ice temperature and then stirred for 5 hours atroom temperature. At room temperature, 2 M hydrochloric acid (120 mL)was added dropwise and stirred for 1 hour. A precipitate was sucked andfiltered and was washed with distilled water (100 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-PAE [20.5 g, yield: about 86.2%, PAE introduction rate:72%, moisture: 2.2%, 247 kDa, Glu D:L ratio (36:64)].

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.2H), 1.4-2.4 (brm, 6.8H),2.6-2.8 (brm, 0.9H), 2.8-3.1 (brm, 2.5H), 3.9-4.5 (brm, 4.7H), 4.5-5.5(brm, 0.2H), 6.9-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-9.1(brm, 2.6H).

Example 6 Synthesis of γ-PGA-PAE

In a 100-mL Scott bottle, distilled water (39 mL) and 1 M NaOH (9.0 mL)were mixed at room temperature. Into this solution, γ-PGA (1.2 g, 65kDa) was dissolved and then ice-cooled. At ice temperature, WSC.HCl (1.8g) was added. After this solution was stirred for 5 minutes, PAE.HCl(1.3 g) was added. This solution was reacted overnight from icetemperature to room temperature and stirred for 20 hours. At roomtemperature, 2 M hydrochloric acid (11 mL) was added dropwise andstirred for 3 hours. A precipitate was sucked and filtered and waswashed with distilled water (20 mL) twice. The precipitate was driedunder reduced pressure at room temperature to acquire γ-PGA-PAE (2.0 g,yield: about 98.8%, PAE introduction rate: 52%, moisture: 2.9%, 89 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.3H), 1.4-2.4 (brm, 7.9H),2.6-2.8 (brm, 0.9H), 2.8-3.1 (brm, 2.6H), 3.9-4.5 (brm, 4.8H), 4.5-5.5(brm, 0.2H), 6.9-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.8H).

Example 7 Synthesis of γ-PGA-PAE

In a 1-L four-necked flask, distilled water (440 mL) and γ-PGA sodiumsalt [15.0 g, 29 kDa, D:L ratio (83:17)] were measured and dissolved atroom temperature and then ice-cooled. At ice temperature, WSC.HCl (19.2g) was added and stirred for 5 minutes. PAE.HCl (13.8 g) was then added.The solution was stirred for 1 hour at ice temperature and then stirredfor 5 hours at room temperature. At room temperature, 2 M hydrochloricacid (120 mL) was added dropwise and stirred for 1 hour. A precipitatewas sucked and filtered and was washed with distilled water (100 mL)twice. The precipitate was dried under reduced pressure at roomtemperature to acquire γ-PGA-PAE [18.2 g, yield: about 91.2%, PAEintroduction rate: 59%, moisture: 3.5%, 56 kDa, Glu D:L ratio (50:50)].

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.0H), 1.4-2.4 (brm, 5.7H),2.6-2.8 (brm, 0.5H), 2.8-3.1 (brm, 2.3H), 3.9-4.5 (brm, 4.2H), 4.5-5.5(brm, 0.2H), 6.9-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.2H).

Example 8 Synthesis of γ-PGA-PAE

In a 1-L four-necked flask, distilled water (440 mL) and γ-PGA sodiumsalt (15.0 g, 29 kDa) were measured and dissolved at room temperatureand then ice-cooled. At ice temperature, WSC.HCl (19.2 g) was added andstirred for 5 minutes. PAE.HCl (13.8 g) was then added. The solution wasstirred for 1.5 hours at ice temperature and then stirred for 20.5 hoursat room temperature. At room temperature, 2 M hydrochloric acid (120 mL)was added dropwise and stirred for 1 hour. A precipitate was sucked andfiltered and was washed with distilled water (100 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-PAE (19.3 g, yield: about 90.9%, PAE introduction rate:70%, moisture: 2.1%, 54 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.4H), 1.4-2.4 (brm, 5.4H),2.6-2.8 (brm, 0.7H), 2.8-3.1 (brm, 2.3H), 3.9-4.5 (brm, 4.0H), 4.5-5.5(brm, 0.1H), 6.9-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.2H).

Example 9 Synthesis of γ-PGA-PAE

In a 20-mL sample bottle, NaHCO₃ (42 mg) was measured and dissolved indistilled water (2.5 mL) at room temperature. After γ-PGA (61 mg, 148kDa) was added and dissolved, the solution was ice-cooled. At icetemperature, DMTMM (453 mg) was added and stirred for 7 minutes. PAE.HCl(108 mg) was then added. The solution was stirred for 1.0 hour at icetemperature and then stirred for about 15 hours at room temperature. Atroom temperature, 2 M hydrochloric acid (0.47 mL) was added dropwise andstirred for 1 hour. A precipitate was sucked and filtered and was washedwith distilled water. The precipitate was dried under reduced pressureat room temperature to acquire γ-PGA-PAE (103 mg, PAE introduction rate:68%).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 2.9H), 1.4-2.4 (brm, 5.9H),2.8-3.1 (brm, 2.0H), 3.9-4.5 (brm, 4.3H), 6.9-7.5 (brm, 5.0H, a relativevalue when the protons of the phenyl group of the phenylalanyl group areassumed as 5.0H), 7.6-8.7 (brm, 2.2H).

Example 10 Synthesis of γ-PGA-D-PAE

In a 100-mL Scott bottle, distilled water (39 mL) and γ-PGA sodium salt[1.62 g, 29 kDa, D:L ratio (83:17)] were measured, dissolved, and thenice-cooled. At ice temperature, WSC.HCl (1.80 g) was added and stirredfor 5 minutes. D-phenylalanine ethyl ester hydrochloride (D-PAE.HCl)(1.30 g) was added and vigorously stirred for 1.3 hours at icetemperature. The solution was then stirred for 5 hours at roomtemperature. At room temperature, 2 M hydrochloric acid (14 mL) wasadded dropwise and stirred for 1.5 hours. A precipitate was sucked andfiltered and was washed with distilled water (10 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-D-PAE [1.90 g, PAE introduction rate: 67%, moisture:2.41%, 64 kDa, Glu D:L ratio (66:34)].

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.2H), 1.4-2.4 (brm, 6.0H),2.6-2.8 (brm, 0.6H), 2.8-3.1 (brm, 2.4H), 3.9-4.5 (brm, 4.4H), 4.5-5.5(brm, 0.2H), 6.9-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.3H).

Example 11 Synthesis of γ-PGA-D-PAE

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA [1.21 g, 65 kDa, D:L ratio (35:65)] was added at roomtemperature and dissolved, the solution was ice-cooled. At icetemperature, WSC.HCl (1.80 g) was added and stirred for 5 minutes.D-PAE.HCl (1.30 g) was added and vigorously stirred for 1.5 hours at icetemperature. The solution was then stirred for 21 hours at roomtemperature. At room temperature, 2 M hydrochloric acid (13 mL) wasadded dropwise and stirred for 2.5 hours. A precipitate was sucked andfiltered and was washed with distilled water (10 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-D-PAE [1.96 g, PAE introduction rate: 56%, moisture:2.35%, 98 kDa, Glu D:L ratio (54:46)].

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.3H), 1.4-2.4 (brm, 7.2H),2.6-2.8 (brm, 0.5H), 2.8-3.1 (brm, 2.4H), 3.9-4.5 (brm, 4.9H), 4.5-5.5(brm, 0.3H), 6.9-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.6H).

Example 12 Synthesis of γ-PGA-DL-PAE

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA [1.21 g, 65 kDa, D:L ratio (35:65)] was added at roomtemperature and dissolved, the solution was ice-cooled. At icetemperature, WSC.HCl (1.80 g) was added and stirred for 5 minutes.DL-PAE.HCl (1.30 g) was added and vigorously stirred for 1.5 hours atice temperature. The solution was then stirred for 21 hours at roomtemperature. At room temperature, 2 M hydrochloric acid (13 mL) wasadded dropwise and stirred for 2.5 hours. A precipitate was sucked andfiltered and was washed with distilled water (10 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-DL-PAE [2.06 g, PAE introduction rate: 56%, moisture:2.51%, 97 kDa, Glu D:L ratio (45:55)].

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.3H), 1.4-2.4 (brm, 7.2H),2.6-2.8 (brm, 0.5H), 2.8-3.1 (brm, 2.5H), 3.9-4.5 (brm, 4.7H), 4.5-5.5(brm, 0.3H), 6.9-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.6H).

Example 13 Synthesis of γ-PGA-PAE

In a 100-mL Scott bottle, NaHCO₃ (0.42 g) and distilled water (49 mL)were measured and sufficiently stirred. After γ-PGA (0.61 g, 114 kDa)was added at room temperature and stirred for 30 minutes and dissolvedat room temperature, the solution was ice-cooled and stirred for 15minutes. At ice temperature, WSC.HCl (0.90 g) was added and stirred for5 minutes. PAE.HCl (1.08 g) was added and vigorously stirred for 1.0hour at ice temperature. The solution was then stirred for 20 hours atroom temperature. At room temperature, 2 M hydrochloric acid (10 mL) wasadded dropwise and stirred for 1 hour. A precipitate was sucked andfiltered and was washed with distilled water (5 mL) twice. Theprecipitate was dried under reduced pressure at 40° C. to acquireγ-PGA-PAE (1.09 g, PAE introduction rate: 67%, moisture: 2.7%).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.1H), 1.4-2.4 (brm, 5.3H),2.6-2.8 (brm, 0.7H), 2.8-3.1 (brm, 2.4H), 3.9-5.5 (brm, 4.4H), 6.9-7.5(brm, 5.0H, a relative value when the protons of the phenyl group of thephenylalanyl group are assumed as 5.0H), 7.6-8.7 (brm, 2.1H).

Example 14 Synthesis of γ-PGA-PAE

In a 50-mL conical flask, NaHCO₃ (0.42 g) and distilled water (36 mL)were measured and sufficiently stirred. After γ-PGA (0.61 g, 114 kDa)was added at room temperature and stirred for 30 minutes and dissolvedat room temperature, the solution was ice-cooled and stirred for 15minutes. At ice temperature, WSC.HCl (0.90 g) was added and stirred for5 minutes. PAE.HCl (1.08 g) was added and vigorously stirred for 1.0hour at ice temperature. The solution was then stirred for 20 hours atroom temperature. At room temperature, 2 M hydrochloric acid (10 mL) wasadded dropwise and stirred for 1 hour. A precipitate was sucked andfiltered and was washed with distilled water (5 mL) twice. Theprecipitate was dried under reduced pressure at 40° C. to acquireγ-PGA-PAE (1.14 g, PAE introduction rate: 67%, moisture: 2.9%).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.2H), 1.4-2.4 (brm, 5.2H),2.6-3.1 (brm, 3.0H), 3.9-5.5 (brm, 4.0H), 6.9-7.5 (brm, 5.0H, a relativevalue when the protons of the phenyl group of the phenylalanyl group areassumed as 5.0H), 7.6-8.7 (brm, 2.1H).

Example 15 Synthesis of γ-PGA-PAE

In a 50-mL conical flask, NaHCO₃ (0.42 g) and distilled water (30 mL)were measured and sufficiently stirred. After γ-PGA (0.61 g, 114 kDa)was added at room temperature and stirred for 30 minutes and dissolvedat room temperature, the solution was ice-cooled and stirred for 15minutes. At ice temperature, WSC.HCl (0.90 g) was added and stirred for5 minutes. PAE.HCl (1.08 g) was added and vigorously stirred for 1.0hour at ice temperature. The solution was then stirred for 20 hours atroom temperature. At room temperature, 2 M hydrochloric acid (10 mL) wasadded dropwise and stirred for 1 hour. A precipitate was sucked andfiltered and was washed with distilled water (5 mL) twice. Theprecipitate was dried under reduced pressure at 40° C. to acquireγ-PGA-PAE (1.18 g, PAE introduction rate: 70%, moisture: 2.3%).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.2H), 1.4-2.4 (brm, 5.1H),2.6-3.1 (brm, 2.9H), 3.9-5.5 (brm, 4.0H), 6.9-7.5 (brm, 5.0H, a relativevalue when the protons of the phenyl group of the phenylalanyl group areassumed as 5.0H), 7.6-8.7 (brm, 2.1H).

Example 16 Synthesis of γ-PGA-PAE

In a 30-mL conical flask, NaHCO₃ (0.42 g) and distilled water (21 mL)were measured and sufficiently stirred. After γ-PGA (0.61 g, 114 kDa)was added at room temperature and stirred for 30 minutes and dissolvedat room temperature, the solution was ice-cooled and stirred for 5minutes. At ice temperature, WSC.HCl (0.90 g) was added and stirred for5 minutes. PAE.HCl (1.08 g) was added and vigorously stirred for 1.0hour at ice temperature. The solution was then stirred for 20 hours atroom temperature. At room temperature, 2 M hydrochloric acid (10 mL) wasadded dropwise and stirred for 1 hour. A precipitate was sucked andfiltered and was washed with distilled water (5 mL) twice. Theprecipitate was dried under reduced pressure at 40° C. to acquireγ-PGA-PAE (1.21 g, PAE introduction rate: 73%, moisture: 2.6%).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.2H), 1.4-2.4 (brm, 5.0H),2.6-2.8 (brm, 0.3H), 2.8-3.1 (brm, 2.3H), 3.9-5.5 (brm, 4.2H), 6.4-6.6(brm, 0.1H), 6.9-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.0H).

Example 17 Synthesis of γ-PGA-PAE

In a 20-mL sample bottle, distilled water (7.8 mL) and 1 M NaOH (0.9 mL)were mixed at room temperature. Into this solution, γ-PGA (121 mg, 239kDa) was dissolved and then ice-cooled. At ice temperature, WSC.HCl (180mg) was added. After stirring for 5 minutes, PAE.HCl (130 mg) was added.A reaction was performed at ice temperature for 1 hour and at roomtemperature for 6 hours. At room temperature, 2 M hydrochloric acid (1.4mL) was added dropwise and stirred for 1.5 hours. A precipitate wassucked and filtered and was washed with distilled water (5 mL) twice.The precipitate was dried under reduced pressure at room temperature toacquire γ-PGA-PAE (185 mg, PAE introduction rate: 50%, moisture: 3.1%,797 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.3H), 1.4-2.4 (brm, 8.0H),2.6-2.8 (brm, 1.0H), 2.8-3.1 (brm, 2.5H), 3.9-4.5 (brm, 5.0H), 4.5-5.5(brm, 0.3H), 6.9-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.8H).

Example 18 Synthesis of α-L-PGA-D-PAE

In a 20-mL sample bottle, α-L-PGA sodium salt (136 mg) and distilledwater (4.8 mL) were measured and dissolved. The solution was thenice-cooled. At ice temperature, WSC.HCl (180 mg) was added and stirredfor 5 minutes. D-PAE.HCl (129 mg) was added and vigorously stirred for1.5 hours at ice temperature. The solution was then stirred for 16 hoursat room temperature. At room temperature, 2 M hydrochloric acid (0.14mL) was added dropwise and stirred for 1.5 hours. A precipitate wassucked and filtered and was washed with distilled water (5 mL) twice.The precipitate was dried under reduced pressure at room temperature toacquire α-L-PGA-D-PAE (206 mg, PAE introduction rate: 70%, moisture:2.20%).

¹H NMR (500 MHz, DMSO-d₆) δ 1.03-1.05 (brm, 3.53H), 1.50-2.35 (brm,5.70H), 2.50-2.60 (brm, 1.24H), 2.60-2.85 (brm, 2.14H), 2.85-3.05 (brm,2.14H), 3.10-3.25 (brm, 0.63H), 3.50-3.80 (brm, 0.83H), 3.90-5.00 (brm,4.68H), 7.04-7.35 (brm, 5H, a relative value when the protons of thephenyl group of the phenylalanyl group are assumed as 5.0H), 7.70-9.30(brm, 2.48H).

Example 19 Synthesis of α-L-PGA-L-PAE

In a 20-mL sample bottle, α-L-PGA sodium salt (136 mg) and distilledwater (4.8 mL) were measured and dissolved. The solution was thenice-cooled. At ice temperature, WSC.HCl (180 mg) was added and stirredfor 5 minutes. L-PAE.HCl (129 mg) was added and vigorously stirred for1.5 hours at ice temperature. The solution was then stirred for 16 hoursat room temperature. At room temperature, 2 M hydrochloric acid (0.14mL) was added dropwise and stirred for 1.5 hours. A precipitate wassucked and filtered and was washed with distilled water (5 mL) twice.The precipitate was dried under reduced pressure at room temperature toacquire α-L-PGA-L-PAE (216 mg, PAE introduction rate: 69%, moisture:2.44%).

¹H NMR (500 MHz, DMSO-d₆) δ 1.05-1.06 (brm, 3.52H), 1.50-2.40 (brm,5.71H), 2.50-2.75 (brm, 1.59H), 2.75-3.05 (brm, 2.41H), 3.10-3.25 (brm,0.75H), 3.50-3.80 (brm, 0.59H), 3.90-4.50 (brm, 4.56H), 4.50-5.58 (brm,0.02H), 7.04-7.35 (brm, 5H, a relative value when the protons of thephenyl group of the phenylalanyl group are assumed as 5.0H), 7.70-9.30(brm, 2.49H).

Example 20 Synthesis of α-D-PGA-D-PAE

In a 20-mL sample bottle, α-D-PGA sodium salt (408 mg) and distilledwater (14.4 mL) were measured and dissolved. The solution was thenice-cooled. At ice temperature, WSC.HCl (540 mg) was added and stirredfor 5 minutes. D-PAE.HCl (387 mg) was added and vigorously stirred for 1hour at ice temperature. The solution was then stirred for 12 hours atroom temperature. At room temperature, 2 M hydrochloric acid (0.5 mL)was added dropwise and stirred for 1.5 hours. A precipitate was suckedand filtered and was washed with distilled water (15 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire α-D-PGA-D-PAE (579 mg, PAE introduction rate: 77%, moisture:0.97%).

¹H NMR (500 MHz, DMSO-d₆) δ 0.98-1.06 (brm, 3.35H), 1.50-2.35 (brm,5.18H), 2.55-2.80 (brm, 0.96H), 2.80-3.05 (brm, 2.31H), 3.05-3.80 (brm,2.72H), 3.90-4.80 (brm, 4.35H), 7.00-7.40 (brm, 5H, a relative valuewhen the protons of the phenyl group of the phenylalanyl group areassumed as 5.0H), 7.70-9.30 (brm, 2.31H).

Example 21 Synthesis of α-D-PGA-L-PAE

In a 20-mL sample bottle, α-D-PGA sodium salt (408 mg) and distilledwater (14.4 mL) were measured and dissolved. The solution was thenice-cooled. At ice temperature, WSC.HCl (540 mg) was added and stirredfor 5 minutes. L-PAE.HCl (387 mg) was added and vigorously stirred for 1hour at ice temperature. The solution was then stirred for 12 hours atroom temperature. At room temperature, 2 M hydrochloric acid (0.5 mL)was added dropwise and stirred for 1.5 hours. A precipitate was suckedand filtered and was washed with distilled water (15 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire α-D-PGA-L-PAE (605 mg, PAE introduction rate: 76%, moisture:1.09%).

¹H NMR (500 MHz, DMSO-d₆) δ 0.98-1.04 (brm, 3.44H), 1.50-2.40 (brm,5.26H), 2.55-2.75 (brm, 1.02H), 2.80-3.05 (brm, 2.32H), 3.05-3.80 (brm,3.00H), 3.90-4.70 (brm, 4.37H), 7.00-7.45 (brm, 5H, a relative valuewhen the protons of the phenyl group of the phenylalanyl group areassumed as 5.0H), 7.70-9.30 (brm, 2.34H).

Example 22 Synthesis of αβ-DL-Poly Asp-D-PAE

In a 20-mL sample bottle, αβ-DL-Poly Asp sodium salt (136 mg) anddistilled water (4.8 mL) were measured and dissolved. The solution wasthen ice-cooled. At ice temperature, WSC.HCl (180 mg) was added andstirred for 5 minutes. D-PAE.HCl (129 mg) was added and vigorouslystirred for 1.5 hours at ice temperature. The solution was then stirredfor 13 hours at room temperature. At room temperature, 2 M hydrochloricacid (0.2 mL) was added and stirred for 1.5 hours. A precipitate wassucked and filtered and was washed with distilled water (5 mL) twice.The precipitate was dried under reduced pressure at room temperature toacquire αβ-DL-Poly Asp-D-PAE (183 mg, PAE introduction rate: about 63%,moisture: 2.37%).

¹H NMR (500 MHz, DMSO-d₆) δ 0.75-1.25 (brm, 3.30H), 1.60-2.80 (brm,3.17H), 2.80-3.75 (brm, 6.28H), 3.75-4.15 (brm, 2.06H), 4.15-5.50 (brm,2.70H), 6.90-7.45 (brm, 5H, a relative value when the protons of thephenyl group of the phenylalanyl group are assumed as 5.0H), 7.60-9.20(brm, 2.35H).

Example 23 Synthesis of αβ-DL-Poly Asp-L-PAE

In a 20-mL sample bottle, αβ-DL-Poly Asp sodium salt (136 mg) anddistilled water (4.8 mL) were measured and dissolved. The solution wasthen ice-cooled. At ice temperature, WSC.HCl (180 mg) was added andstirred for 5 minutes. L-PAE.HCl (129 mg) was added and vigorouslystirred for 1.5 hours at ice temperature. The solution was then stirredfor 16 hours at room temperature. At room temperature, 2 M hydrochloricacid (0.2 mL) was added and stirred for 1.5 hours. A precipitate wassucked and filtered and was washed with distilled water (5 mL) twice.The precipitate was dried under reduced pressure at room temperature toacquire αβ-DL-Poly Asp-L-PAE (176 mg, PAE introduction rate: about 62%,moisture: 2.68%).

¹H NMR (500 MHz, DMSO-d₆) δ 0.80-1.30 (brm, 3.23H), 1.60-2.80 (brm,3.22H), 2.80-3.80 (brm, 6.41H), 3.80-4.20 (brm, 2.08H), 4.20-5.30 (brm,2.75H), 6.90-7.45 (brm, 5H, a relative value when the protons of thephenyl group of the phenylalanyl group are assumed as 5.0H), 7.60-9.30(brm, 2.35H).

Comparison Example 1 Synthesis of γ-PGA-PAE by Desalting Method

In a 200-mL conical flask, NaHCO₃ (0.42 g) and distilled water (100 mL)were measured and sufficiently stirred. After γ-PGA (0.61 g) was addedat room temperature and stirred for 30 minutes and dissolved at roomtemperature, the solution was ice-cooled and stirred for 15 minutes. Atice temperature, WSC.HCl (0.90 g) was added and stirred for 5 minutes.PAE.HCl (1.08 g) was added and vigorously stirred for 1.0 hour at icetemperature. The solution was then stirred for 22 hours at roomtemperature. Subsequently, a desalting membrane (Wako, Spectra/Por®132124, 15 kDa cutoff) was used for desalting (5 L of water was changedtwice for 3 days). Acquired retained liquid (about 200 mL) was frozenand then subjected to lyophilization to acquire γ-PGA-PAE (1.039 g,moisture: 5.85%). EtOH (80 mL) was added and shaken at room temperature(200 rpm, 2 hours). After centrifugal separation (4500 rpm, 30 minutes,5° C.), supernatant was removed by decantation. This was dried underreduced pressure was conducted at room temperature to acquire γ-PGA-PAE(0.97 g, PAE introduction rate: 48%, moisture: 1.85%).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 4.0H), 1.4-2.4 (brm, 10.1H),2.6-2.8 (brm, 0.6H), 2.8-3.1 (brm, 3.2H), 3.35-3.6 (q, 0.6H), 3.9-5.5(brm, 5.3H), 5.7-6.0 (0.3H), 6.9-7.5 (brm, 5.0H, a relative value whenthe protons of the phenyl group of the phenylalanyl group are assumed as5.0H), 7.6-8.7 (brm, 2.6H).

Example 24 Synthesis of Graft Copolymer (γ-PGA-Phe-OBn) ofpoly(γ-glutamic acid) and phenylalanine benzyl ester

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. L-phenylalanine benzylester hydrochloride (L-H-Phe-OBn.HCl) (1.1 g) was added and vigorouslystirred for 1 hour at ice temperature. The solution was then stirred for17.5 hours at room temperature. At room temperature, 2 M hydrochloricacid (15 mL) was added dropwise and stirred for 1.5 hours. A precipitatewas sucked and filtered and was washed with distilled water (50 mL). Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-Phe-OBn (1.95 g, yield: about 74.2%. Phe-OBn introductionrate: 63%, moisture: 4.67%, 72 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.2 (brm, 0.45H), 1.3-2.35 (brm, 6.52H),2.6-2.8 (brm, 1.06H), 2.8-3.2 (brm, 2.16H), 4.0-4.4 (brm, 1.56H),4.4-4.6 (brs, 0.62H), 4.6-4.9 (brm, 0.26H), 4.9-5.2 (brm, 1.06H),6.9-7.5 (brm, 5.0H, a relative value when the protons of the phenylgroup of the phenylalanyl group are assumed as 5.0H), 7.7-9.0 (brm,1.83H).

Example 25 Synthesis of γ-PGA-Phe-OBn

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. H-Phe-OBn.HCl (0.55 g) wasadded and vigorously stirred for 1 hour at ice temperature. The solutionwas then stirred for 17.5 hours at room temperature. At roomtemperature, 2 M hydrochloric acid (15 mL) was added dropwise andstirred for 1.5 hours. A precipitate was sucked and filtered and waswashed with distilled water (50 mL). The precipitate was dried underreduced pressure at room temperature to acquire γ-PGA-Phe-OBn (1.39 g,yield: about 72.6%, Phe-OBn introduction rate: 30%, moisture: 5.57%, 31kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 1.4-2.3 (brm, 13.99H), 2.6-2.8 (brm, 2.72H),2.8-3.2 (brm, 4.06H), 4.0-4.49 (brm, 3.70H), 4.9-5.2 (brm, 1.33H),7.0-7.5 (brm, 5.0H, a relative value when the protons of the phenylgroup of the phenylalanyl group are assumed as 5.0H), 7.7-9.1 (brm,3.90H).

Example 26 Synthesis of Graft Copolymer (γ-PGA-Phe-OcPen) ofpoly(γ-glutamic acid) and phenylalanine cyclopentyl ester

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. After (2S)-phenylalaninecyclopentyl ester hydrochloride [(2S)—H-Phe-OcPen.HCl] (1.52 g) wasadded, the solution was vigorously stirred for 1 hour at icetemperature. The solution was then stirred for 14 hours at roomtemperature. At room temperature, 2 M hydrochloric acid (13 mL) wasadded dropwise and stirred for 1 hour. A precipitate was sucked andfiltered and was washed with distilled water (10 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-Phe-OcPen (2.33 g, yield: about 96.8%, Phe-OcPenintroduction rate: 57%, moisture: 2.88%, 282 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.2 (brm, 0.43H), 1.3-2.3 (brm, 14.97H),2.3-2.8 (brm, 1.60H), 2.8-3.1 (brm, 2.25H), 4.0-4.4 (brm, 2.65H),4.4-4.9 (brs, 0.24H), 4.9-5.2 (brm, 1.06H), 7.0-7.4 (brm, 5.0H, arelative value when the protons of the phenyl group of the phenylalanylgroup are assumed as 5.0H), 7.7-9.2 (brm, 2.57H).

Example 27 Synthesis of γ-PGA-Phe-OcPen

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. After (2S)-phenylalaninecyclopentyl ester hydrochloride [(2S)—H-Phe-OcPen.HCl] (1.01 g) wasadded, the solution was vigorously stirred for 1 hour at icetemperature. The solution was then stirred for 14 hours at roomtemperature. At room temperature, 2 M hydrochloric acid (13 mL) wasadded dropwise and stirred for 1 hour. A precipitate was sucked andfiltered and was washed with distilled water (10 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-Phe-OcPen (1.89 g, yield: about 95.8%, Phe-OcPenintroduction rate: 36%, moisture: 3.51%, 93 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.2 (brm, 0.55H), 1.3-2.3 (brm, 19.20H),2.6-2.8 (brm, 1.48H), 2.8-3.1 (brm, 2.94H), 4.0-4.4 (brm, 3.77H),4.4-5.2 (brm, 1.55H), 7.0-7.4 (brm, 5.0H, a relative value when theprotons of the phenyl group of the phenylalanyl group are assumed as5.0H), 7.8-9.2 (brm, 3.45H).

Example 28 Synthesis of Graft Copolymer (γ-PGA-Phe-OtBu) ofpoly(γ-glutamic acid) and phenylalanine t-butyl ester

In a 100-mL Scott bottle, 1M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. L-phenylalanine t-butylester hydrochloride (L-H-Phe-OtBu.HCl) (1.94 g) was added and vigorouslystirred for 1 hour at ice temperature. The solution was then stirred for19 hours at room temperature. At room temperature, 2 M hydrochloric acid(13 mL) was added dropwise and stirred for 1 hour. A precipitate wassucked and filtered and was washed with distilled water (10 mL) twice.The precipitate was dried under reduced pressure at room temperature toacquire γ-PGA-Phe-OtBu (3.39 g, yield: about 98.2%, Phe-OtBuintroduction rate: 78%, moisture: 25.04%, 106 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.5 (brm, 9.31H), 1.5-2.3 (brm, 5.27H),2.72 (brm, 0.68H), 2.8-3.1 (brm, 2.31H), 4.1-4.5 (brm, 2.09H), 4.5-5.1(brm, 0.05H), 7.0-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.7-9.8(brm, 2.24H).

Example 29 Synthesis of γ-PGA-Phe-OtBu

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. L-phenylalanine t-butylester hydrochloride (L-H-Phe-OtBu.HCl) (1.45 g) was added and vigorouslystirred for 1 hour at ice temperature. The solution was then stirred for19 hours at room temperature. At room temperature, 2 M hydrochloric acid(13 mL) was added dropwise and stirred for 1 hour. A precipitate wassucked and filtered and was washed with distilled water (10 mL) twice.The precipitate was dried under reduced pressure at room temperature toacquire γ-PGA-Phe-OtBu (3.28 g, yield: about 100.0%, Phe-OtBuintroduction rate: 57%, moisture: 29.72%, 71 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.5 (brm, 9.31H), 1.5-2.3 (brm, 5.27H),2.72 (brm, 0.68H), 2.8-3.1 (brm, 2.31H), 4.1-4.5 (brm, 2.09H), 4.4-5.1(brm, 0.05H), 7.0-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.7-9.8(brm, 2.24H).

Example 30 Synthesis of Graft Copolymer (γ-PGA-Phe-OMe) ofpoly(γ-glutamic acid) and phenylalanine methyl ester

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. L-phenylalanine methylester hydrochloride (L-H-Phe-OMe.HCl) (1.62 g) was added and vigorouslystirred for 1.5 hours at ice temperature. The solution was then stirredfor 15 hours at room temperature. At room temperature, 2 M hydrochloricacid (14 mL) was added dropwise and stirred for 1 hour. A precipitatewas sucked and filtered and was washed with distilled water (10 mL)thrice. The precipitate was dried under reduced pressure at roomtemperature to acquire γ-PGA-Phe-OMe (2.24 g, yield: about 99.6%,Phe-OMe introduction rate: 72%, moisture: 4.78%, 260 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 1.4-2.4 (brm, 5.53H), 2.4-2.7 (brm, 0.50H),2.8-3.1 (brm, 2.49H), 3.5-3.7 (brm, 3.00H), 4.0-4.6 (brm, 2.36H),4.6-5.1 (brm, 0.19H), 6.9-7.5 (brm, 5.0H, a relative value when theprotons of the phenyl group of the phenylalanyl group are assumed as5.0H), 7.7-9.2 (brm, 2.25H).

Example 31 Synthesis of γ-PGA-Phe-OMe

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. L-phenylalanine methylester hydrochloride (L-H-Phe-OMe.HCl) (1.22 g) was added and vigorouslystirred for 1.5 hours at ice temperature. The solution was then stirredfor 15 hours at room temperature. At room temperature, 2 M hydrochloricacid (14 mL) was added dropwise and stirred for 1 hour. A precipitatewas sucked and filtered and was washed with distilled water (10 mL)thrice. The precipitate was dried under reduced pressure at roomtemperature to acquire γ-PGA-Phe-OMe (2.07 g, yield: about 97.6%,Phe-OMe introduction rate: 53%, moisture: 4.70%, 205 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 1.4-2.4 (brm, 7.61H), 2.5-2.7 (brm, 0.80H),2.8-3.1 (brm, 2.92H), 3.5-3.7 (brm, 3.00H), 4.0-4.6 (brm, 2.92H),4.6-5.2 (brm, 0.31H), 6.9-7.5 (brm, 5.0H, a relative value when theprotons of the phenyl group of the phenylalanyl group are assumed as5.0H), 7.7-9.2 (brm, 2.71H).

Example 32 Synthesis of Graft Copolymer (γ-PGA-Phe-OH) ofpoly(γ-glutamic acid) and phenylalanine

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. A hydrochloric acidaqueous solution (13.2 mL) of L-phenylalanine (L-H-Phe-OH) (1.24 g) wasadded and vigorously stirred for 1 hour at ice temperature. The solutionwas then stirred for 14 hours at room temperature. At room temperature,2 M hydrochloric acid (12 mL) was added dropwise and stirred for 2hours. A precipitate was taken out and washed with distilled water (25mL) twice. The precipitate was dried under reduced pressure at roomtemperature to acquire γ-PGA-Phe-OH (0.26 g, yield: about 17.3%, Phe-OHintroduction rate: 19%, moisture: 7.11%, 271 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 1.5-2.4 (brm, 21.68H), 2.4-2.8 (brm, 3.09H),2.8-3.2 (brm, 5.61H), 4.0-5.2 (brm, 8.09H), 5.3-6.2 (brm, 0.27), 6.9-7.4(brm, 5.0H, a relative value when the protons of the phenyl group of thephenylalanyl group are assumed as 5.0H), 7.7-8.5 (brm, 4.98H).

Example 33 Synthesis of Graft Copolymer (γ-PGA-Phe-NH₂) ofpoly(γ-glutamic acid) and phenylalaninamide

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. A hydrochloric acidaqueous solution (12 mL) of L-phenylalaninamide (L-H-Phe-NH₂) (1.24 g)was added and vigorously stirred for 1 hour at ice temperature. Thesolution was then stirred for 16 hours at room temperature. At roomtemperature, 2 M hydrochloric acid (12 mL) was added dropwise andstirred for 2 hours. A precipitate was taken out and washed withdistilled water (30 mL) twice. The precipitate was dried under reducedpressure at room temperature to acquire γ-PGA-Phe-NH₂ (1.043 g, yield:about 63.3%, Phe-NH₂ introduction rate: 26%, moisture: 8.79%, 30 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 1.4-2.4 (brm, 20.81H), 2.4-2.8 (brm, 3.57H),2.8-3.2 (brm, 7.33H), 4.0-5.2 (brm, 8.82H), 6.9-7.3 (brm, 6.57H, arelative value when 5.0H of the protons of the phenyl group of thephenylalanyl group is assumed as 6.57H), 7.3-7.7(1.09), 7.7-8.5 (brm,5.35H).

Example 34 Synthesis of Graft Copolymer (γ-PGA-p-F-Phe-OEt) ofpoly(γ-glutamic acid) and 4-fluorophenylalanine ethyl ester

In a 100-mL Scott bottle, 1M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. L-4-fluorophenylalanineethyl ester hydrochloride (L-H-p-F-Phe-OEt.HCl) (1.40 g) was added andvigorously stirred for 1 hour at ice temperature. The solution was thenstirred for 12 hours at room temperature. At room temperature, 2 Mhydrochloric acid (13 mL) was added dropwise and stirred for 1 hour. Aprecipitate was sucked and filtered and was washed with distilled water(50 mL) twice. The precipitate was dried under reduced pressure at roomtemperature to acquire γ-PGA-p-F-Phe-OEt (2.22 g, yield: about 96.2%,p-F-Phe-OEt introduction rate: 56%, moisture: 3.25%, 325 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.8-1.8 (brm, 3.38H), 1.4-2.3 (brm, 7.10),2.4-2.7 (brm, 1.18), 2.8-3.1 (brm, 2.55H), 3.9-4.5 (brm, 4.78H), 4.5-5.2(brm, 0.27H), 6.8-7.5 (brm, 4.00H; a relative value when the protons ofthe phenyl group are assumed as 4.0H), 7.7-9.2 (brm, 2.59H).

Example 35 Synthesis of Graft Copolymer (γ-PGA-p-Cl-Phe-OEt) ofpoly(γ-glutamic acid) and 4-chlorophenylalanine ethyl ester

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. DL-4-chlorophenylalanineethyl ester hydrochloride (DL-H-p-Cl-Phe-OEt.HCl) (1.49 g) was added andvigorously stirred for 1 hour at ice temperature. The solution was thenstirred for 15.5 hours at room temperature. At room temperature, 2 Mhydrochloric acid (14 mL) was added dropwise and stirred for 1 hour. Aprecipitate was sucked and filtered and was washed with distilled water(20 mL) twice. The precipitate was dried under reduced pressure at roomtemperature to acquire γ-PGA-p-Cl-Phe-OEt (2.26 g, yield: about 96.6%,p-Cl-Phe-OEt introduction rate: 58%, moisture: 3.65% 284 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 1.0-1.3 (brm, 3.30H). 1.4-2.4 (brm, 6.95),2.4-2.7 (brm, 1.19), 2.8-3.1 (brm, 2.68H), 3.9-4.5 (brm, 4.70H), 4.5-5.2(brm, 0.20H), 7.0-7.5 (brm, 4.00H; a relative value when the protons ofthe phenyl group are assumed as 4.0H), 7.7-9.2 (brm, 2.57H).

Example 36 Synthesis of Graft Copolymer (γ-PGA-p-Br-Phe-OEt) ofpoly(γ-glutamic acid) and 4-bromophenylalanine ethyl ester

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. (S)-4-bromophenylalanineethyl ester hydrochloride [(S)—H-p-Br-Phe-OEt.HCl] (1.74 g) was addedand vigorously stirred for 1 hour at ice temperature. The solution wasthen stirred for 13 hours at room temperature. At room temperature, 2 Mhydrochloric acid (13 mL) was added dropwise and stirred for 1 hour. Aprecipitate was sucked and filtered and was washed with distilled water(20 mL) twice. The precipitate was dried under reduced pressure at roomtemperature to acquire γ-PGA-p-Br-Phe-OEt (2.43 g, yield: about 97.2%,p-Br-Phe-OEt introduction rate: 56%, moisture: 2.39%, 185 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brm, 3.34H), 1.4-2.4 (brm, 7.17),2.4-2.7 (brm, 1.46), 2.7-3.1 (brm, 2.76H), 3.9-4.5 (brm, 4.90H), 4.5-5.2(brm, 0.33H), 6.9-7.3 (brm, 4.00H; 2.0H was defined as a relative valueout of 4.0H for the protons of the phenyl group), 7.3-7.7 (brm, 2.0H),7.7-9.2 (brm, 2.63H).

Example 37 Synthesis of Graft Copolymer (γ-PGA-p-NO₂-Phe-OEt) ofpoly(γ-glutamic acid) and 4-nitrophenylalanine ethyl ester

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. D-p-nitrophenylalanineethyl ester hydrochloride (D-H-p-NO₂-Phe-OEt.HCl) (1.55 g) was added andvigorously stirred for 1 hour at ice temperature. The solution was thenstirred for 12 hours at room temperature. At room temperature, 2 Mhydrochloric acid (13 mL) was added dropwise and stirred for 1 hour. Aprecipitate was sucked and filtered and was washed with distilled water(50 mL) twice. The precipitate was dried under reduced pressure at roomtemperature to acquire γ-PGA-p-NO₂-Phe-OEt (2.39 g, yield: about 95.7%,p-NO₂-Phe-OEt introduction rate: 58%, moisture: 3.42%, 331 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.8-1.3 (brm, 3.28H), 1.4-2.4 (brm, 6.90),2.4-2.7 (brm, 1.06), 2.7-3.3 (brm, 3.76H), 3.9-5.1 (brm, 1.25H), 7.3-7.6(brm, 2.00H; 2.0H was defined as a relative value out of 4.0H for theprotons of the phenyl group), 7.6-9.2 (brm, 0.03H).

Example 38 Synthesis of Graft Copolymer (γ-PGA-p-OiPr-Phe-OEt) ofpoly(γ-glutamic acid) and O-(1-methylethyl)-L-tyrosine ethyl ester

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes.O-(1-methylethyl)-L-tyrosine ethyl ester hydrochloride(L-H-p-OiPr-Phe-OEt.HCl) (1.62 g) was added and vigorously stirred for 1hour at ice temperature. The solution was then stirred for 14 hours atroom temperature. At room temperature, 2 M hydrochloric acid (13 mL) wasadded dropwise and stirred for 1 hour. A precipitate was sucked andfiltered and was washed with distilled water (10 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-p-OiPr-Phe-OEt (2.43 g, yield: about 95.6%, p-OiPr-Phe-OEtintroduction rate: 57%, moisture: 2.82%, 309 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brm, 9.58H), 1.5-2.4 (brm, 7.11),2.6-2.8 (brm, 0.85), 2.8-3.1 (brm, 2.52H), 3.9-4.9 (brm, 5.92H), 6.78(brs, 2.0H; 2.0H was defined as a relative value out of 4.0H for theprotons of the phenyl group), 7.0-7.1 (brm, 2.0H), 7.8-9.2 (brm, 2.76H).

Example 39 Synthesis of Graft Copolymer (γ-PGA-α-Phegly-OEt) ofpoly(γ-glutamic acid) and α-phenylglycine ethyl ester

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. D-α-phenylglycine ethylester hydrochloride (D-H-α-Phegly-OEt.HCl) (1.22 g) was added andvigorously stirred for 1 hour at ice temperature. The solution was thenstirred for 13 hours at room temperature. At room temperature, 2 Mhydrochloric acid (13 mL) was added dropwise and stirred for 1 hour. Aprecipitate was sucked and filtered and was washed with distilled water(20 mL) twice. The precipitate was dried under reduced pressure at roomtemperature to acquire γ-PGA-α-Phegly-OEt (2.00 g, yield: about 96.1%,α-Phegly-OEt introduction rate: 59%, moisture: 3.18%, 126 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brm, 3.23H), 1.5-2.3 (brm, 6.84H),2.4-2.7 (brm, 1.21H), 2.7-3.0 (brm, 0.41H), 4.0-4.2 (brs, 2.61H),4.3-4.5 (brm, 1.03H), 4.5-5.2 (brs, 0.19H), 5.3-5.4 (brm, 0.99H),7.0-7.6 (brm, 5.0H, a relative value when the protons of the phenylgroup are assumed as 5.0H), 7.8-9.2 (brm, 2.56H).

Example 40 Synthesis of Graft Copolymer (γ-PGA-Leu-OEt) ofpoly(γ-glutamic acid) and leucine ethyl ester

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. L-leucine ethyl esterhydrochloride (L-H-Leu-OEt.HCl) (1.47 g) was added and vigorouslystirred for 1 hour at ice temperature. The solution was then stirred for6 hours at room temperature. At room temperature, 2 M hydrochloric acid(13 mL) was added dropwise and stirred for 2 hours. A precipitate wastaken out and washed with distilled water (20 mL) twice. The precipitatewas dried under reduced pressure at room temperature to acquireγ-PGA-Leu-OEt (2.25 g, Leu-OEt introduction rate: 48%, moisture: 5.13%,284 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.7-1.0 (brm, 6.5H, indicated as a relativevalue), 1.1-1.3 (brm, 1.0H), 1.4-2.4 (brm, 10.1H), 2.6-2.8 (brm, 1.2H),2.9-3.3 (brm, 1.3H), 3.9-5.2 (brm, 4.7H), 7.5-9.0 (brm, 2.7H).

Example 41 Synthesis of Graft Copolymer (γ-PGA-Ile-OMe) ofpoly(γ-glutamic acid) and isoleucine methyl ester

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. L-isoleucine methyl esterhydrochloride (L-H-Ile-OMe.HCl) (1.47 g) was added and vigorouslystirred for 1 hour at ice temperature. The solution was then stirred for6 hours at room temperature. At room temperature, 2 M hydrochloric acid(13 mL) was added dropwise and stirred for 2 hours. A precipitate wastaken out and washed with distilled water (20 mL) thrice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-Ile-OMe (2.25 g, Ile-OMe introduction rate: 55%, moisture:15.11%, 209 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.7-0.9 (brm, 6.0H), 1.0-1.3 (brm, 1.3H),1.3-1.5 (brm, 1.0H), 1.6-2.4 (brm, 7.8H), 2.6-2.9 (brm, 0.8H), 2.9-3.3(brm, 1.0H), 3.5-3.7 (brm, 3.0H, a relative value when the methyl groupof CO₂Me is assumed as 3.0H), 4.0-5.2 (brm, 2.7H), 7.8-9.8 (brm, 2.7H).

Example 42 Synthesis of Graft Copolymer[γ-PGA-Cys(Bn)-OEt]poly(γ-glutamic acid) and S-benzylcysteine ethylester

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. L-S-benzylcysteine ethylester hydrochloride [L-H-Cys(Bn)-OEt.HCl] (1.56 g) was added andvigorously stirred for 1 hour at ice temperature. The solution was thenstirred for 17 hours at room temperature. At room temperature, 2 Mhydrochloric acid (14 mL) was added dropwise and stirred for 1 hour. Aprecipitate was sucked and filtered and was washed with distilled water(10 mL) twice. The precipitate was dried under reduced pressure at roomtemperature to acquire γ-PGA-Cys(Bn)-OEt [1.36 g, Cys(Bn)-OEtintroduction rate; 60%, moisture: 3.40%, 404 kDa].

¹H NMR (500 MHz, DMSO-d₆) δ 1.0-1.2 (brs, 3.3H), 1.4-2.4 (brm, 6.7H),2.4-2.7 (brm, 0.3H), 2.6-2.8 (brm, 2.6H), 2.8-3.1 (brm, 0.6H), 4.0-4.5(brm, 4.7H), 4.5-5.2 (brm, 0.3H), 7.1-7.5 (brm, 5.0H, a relative valuewhen the protons of the phenyl group are assumed as 5.0H), 7.7-9.2 (brm,2.6H).

Example 43 Synthesis of Graft Copolymer (γ-PGA-Trp-OEt) ofpoly(γ-glutamic acid) and tryptophan ethyl ester

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes. L-tryptophan ethyl esterhydrochloride (L-H-Trp-OEt.HCl) (1.52 g) was added and vigorouslystirred for 1.5 hours at ice temperature. The solution was then stirredfor 22 hours at room temperature. At room temperature, 2 M hydrochloricacid (13 mL) was added dropwise and stirred for 1 hour. A precipitatewas sucked and filtered and was washed with distilled water (10 mL)twice. The precipitate was dried under reduced pressure at roomtemperature to acquire γ-PGA-Trp-OEt (2.33 g, Trp-OEt introduction rate:56%, moisture: 2.97%, 217 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.8-1.2 (brs, 3.4H), 1.5-2.4 (brm, 6.9H),2.5-2.8 (brm, 1.0H), 2.9-3.3 (brm, 3.2H), 3.9-5.2 (brm, 4.7H), 6.8-7.6(brm, 5.0H, a relative value when the protons of the indole group exceptNH are assumed as 5.0H), 7.7-9.5 (brm, 2.6H).

Example 44 Synthesis of γ-PGA-Trp-OEt

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (9.0 mL)and distilled water (35 mL) were measured and sufficiently stirred.After γ-PGA (1.21 g, 148 kDa) was added at room temperature anddissolved, the solution was ice-cooled. At ice temperature, WSC.HCl(1.80 g) was added and stirred for 5 minutes, L-tryptophan ethyl esterhydrochloride (L-H-Trp-OEt.HCl) (1.01 g) was added and vigorouslystirred for 1.5 hours at ice temperature. The solution was then stirredfor 22 hours at room temperature. At room temperature, 2 M hydrochloricacid (13 mL) was added dropwise and stirred for 1 hour. A precipitatewas sucked and filtered and was washed with distilled water (10 mL)twice. The precipitate was dried under reduced pressure at roomtemperature to acquire γ-PGA-Trp-OEt (1.92 g, Trp-OEt introduction rate:33%, moisture: 3.32%, 47 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.8-1.2 (brs, 3.6H), 1.5-2.4 (brm, 10.2H),2.6-2.8 (brm, 1.7H), 2.9-3.3 (brm, 3.9H), 3.9-5.2 (brm, 6.0H), 6.9-7.6(brm, 5.0H, a relative value when the protons of the indole group exceptNH are assumed as 5.0H), 7.8-9.3 (brm, 3.3H).

Example 45 Synthesis of γ-PGA-PAE

In a 1-L four-necked flask, Na₂CO₃ (1.59 g) and distilled water (500 mL)were measured and sufficiently stirred. γ-PGA (3.64 g) was added at roomtemperature and washed with distilled water (50 mL). After stirring atroom temperature for dissolution, the solution was ice-cooled. At icetemperature, WSC.HCl (5.41 g) was added and stirred for 5 minutes.PAE.HCl (6.48 g) was added and vigorously stirred for 2.0 hours at icetemperature. The solution was then stirred for 20 hours at roomtemperature. At room temperature, 2 M hydrochloric acid (45 mL) wasadded dropwise and stirred for 1.5 hours. A precipitate was sucked andfiltered and was washed with distilled water (50 mL) thrice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-PAE (5.83 g, PAE introduction rate: 48%).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.2H), 1.4-2.4 (brm, 7.4H),2.6-2.8 (brm, 0.9H), 2.8-3.1 (brm, 2.9H), 3.9-5.5 (brm, 5.3H), 6.9-7.5(brm, 5.0H, a relative value when the protons of the phenyl group of thephenylalanyl group are assumed as 5.0H), 7.6-8.7 (brm, 2.5H).

Example 46 Synthesis of γ-PGA-PAE

In a 100-mL Scott bottle, 1 M sodium hydroxide aqueous solution (4.5 mL)and distilled water (20 mL) were measured and sufficiently stirred.After γ-PGA (0.61 g) was added at room temperature and dissolved, thesolution was ice-cooled. At ice temperature, WSC.HCl (0.90 g) was addedand stirred for 5 minutes. PAE.HCl (0.65 g) was added and vigorouslystirred for 2.0 hours at ice temperature. The solution was then stirredfor 21 hours at room temperature. 1 M sodium hydroxide aqueous solution(1.9 mL) was then added dropwise and stirred for 0.5 hours.Subsequently, 2 M HCl (13 mL) was added dropwise and stirred for 1 h. Aprecipitate was sucked and filtered and was washed with distilled water(10 mL) twice. The precipitate was dried under reduced pressure at roomtemperature to acquire γ-PGA-PAE (0.99 g, PAE introduction rate: 57%).

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.3H), 1.4-2.4 (brm, 7.1H),2.8-3.1 (brm, 2.5H), 3.9-5.5 (brm, 5.1H), 6.9-7.5 (brm, 5.0H, a relativevalue when the protons of the phenyl group of the phenylalanyl group areassumed as 5.0H), 7.6-9.2 (brm, 2.6H).

Example 47 Preparation of γ-PGA-PAE (28% Na Salt)

γ-PGA-PAE (200 mg, PAE introduction rate: 55%) and DMSO (1.5 mL) weremeasured and stirred at room temperature for 1 hour to acquire asolution. This solution (1.5 mL) was added dropwise into distilled water(28.5 mL). A 0.1 M NaOH aqueous solution (3.1 mL) was added dropwisewith stirring at room temperature to adjust pH to 10.0. Subsequently,0.01 M HCl (0.05 mL) was added with stirring at room temperature. Thissolution was frozen and then subjected to lyophilization to acquireγ-PGA-PAE (28% Na salt) (207 mg, PAE introduction rate: 58%, Na: 42000ppm, Cl: 2271 ppm).

¹H NMR (500 MHz, DMSO-d₆) δ 0.6-1.2 (brs, 2.8H), 1.4-2.4 (brm, 6.6H),2.8-3.1 (brm, 2.7H), 3.8-4.5 (brm, 4.1H), 4.5-5.5 (brm, 0.5H), 6.4-6.6(brm, 0.2H), 6.7-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.1H).

Example 48 Preparation of γ-PGA-PAE (26% Na Salt)

γ-PGA-PAE (200 mg, PAE introduction rate: 55%) and DMSO (1.5 mL) weremeasured and stirred at room temperature for 1 hour to acquire asolution. This solution (1.5 mL) was added dropwise into distilled water(28.5 mL). A 0.1 M NaOH aqueous solution (2.8 mL) was added dropwisewith stirring at room temperature to adjust pH to 8.0. Subsequently,0.01 M HCl (0.02 mL) was added with stirring at room temperature. Thissolution was frozen and then subjected to lyophilization to acquireγ-PGA-PAE (26% Na salt) (210 mg, PAE introduction rate: 56%, Na: 35000ppm, Cl: 15662 ppm).

¹H NMR (500 MHz, DMSO-d₆) δ 0.6-1.2 (brs, 2.8H), 1.4-2.4 (brm, 6.8H),2.8-3.1 (brm, 2.7H), 3.8-4.5 (brm, 4.1H), 4.5-5.5 (brm, 0.5H), 6.4-6.6(brm, 0.2H), 6.7-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.0H).

Example 49 Preparation of γ-PGA-PAE (14% Na Salt)

γ-PGA-PAE (200 mg, PAE introduction rate: 55%) and DMSO (1.5 mL) weremeasured and stirred at room temperature for 1 hour to acquire asolution. This solution (1.5 mL) was added dropwise into distilled water(28.5 mL). A 0.1 M NaOH aqueous solution (2.3 mL) was added dropwisewith stirring at room temperature to adjust pH to 6.0. Subsequently,0.01 M HCl (0.01 mL) was added with stirring at room temperature. Thissolution was frozen and then subjected to lyophilization to acquireγ-PGA-PAE (14% Na salt) (217 mg, PAE introduction rate: 53%, Na: 22000ppm, Cl: 13282 ppm).

¹H NMR (500 MHz, DMSO-d₆) δ 0.6-1.2 (brs, 2.9H), 1.4-2.4 (brm, 7.0H),2.8-3.1 (brm, 2.4H), 3.8-4.5 (brm, 3.9H), 4.5-5.5 (brm, 0.3H), 6.4-6.6(brm, 0.2H), 6.7-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 1.6H).

Example 50 Preparation of γ-PGA-PAE (0% Na Salt)

γ-PGA-PAE (200 mg, PAE introduction rate: 55%) and DMSO (1.5 mL) weremeasured and stirred at room temperature for 1 hour to acquire asolution. This solution (1.5 mL) was added dropwise into distilled water(28.5 mL). A 0.1 M NaOH aqueous solution (0.9 mL) was added dropwisewith stirring at room temperature to adjust pH to 4.0. Subsequently,0.01 M HCl (0.02 mL) was added with stirring at room temperature. Thissolution was frozen and then subjected to lyophilization to acquireγ-PGA-PAE (0% Na salt) (244 mg, PAE introduction rate: 51%, Na: 6900ppm, Cl: 13785 ppm).

¹H NMR (500 MHz, DMSO-d₆) δ 0.6-1.2 (brs, 2.8H), 1.4-2.4 (brm, 6.6H),2.8-3.1 (brm, 2.3H), 3.8-4.5 (brm, 3.8H), 4.5-5.5 (brm, 0.3H), 6.4-6.6(brm, 0.3H), 6.7-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 1.4H).

Example 51 Preparation of γ-PGA-PAE (27% K Salt)

γ-PGA-PAE (200 mg, PAE introduction rate: 55%) and DMSO (1.5 mL) weremeasured and stirred at room temperature for 1 hour to acquire asolution. This solution (1.5 mL) was added dropwise into distilled water(28.5 mL). A0.1 M KOH aqueous solution (3.1 mL) was added dropwise withstirring at room temperature to adjust pH to 10.0. Subsequently, 0.01 MHCl (0.02 mL) was added with stirring at room temperature. This solutionwas frozen and then subjected to lyophilization to acquire γ-PGA-PAE(27% K salt) (215 mg, PAE introduction rate: 53%, K: 62000 ppm, Cl:22769 ppm, 105 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.6-1.2 (brs, 2.7H), 1.4-2.4 (brm, 7.0H),2.8-3.1 (brm, 2.3H), 3.8-4.5 (brm, 3.9H), 4.5-5.5 (brm, 0.3H), 6.4-6.6(brm, 0.3H), 6.7-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.4H).

Example 52 Preparation of γ-PGA-PAE (55% K Salt)

γ-PGA-PAE (200 mg, PAE introduction rate: 55%) and DMSO (1.5 mL) weremeasured and stirred at room temperature for 1 hour to acquire asolution. This solution (1.5 mL) was added dropwise into distilled water(28.5 mL). A 0.1 M KOH aqueous solution (2.8 mL) was added dropwise withstirring at room temperature to adjust pH to 8.0. Subsequently, 0.01 MHCl (0.02 mL) was added with stirring at room temperature. This solutionwas frozen and then subjected to lyophilization to acquire γ-PGA-PAE(55% K salt) (215 mg, PAE introduction rate: 48%, K: 42000 ppm, Cl:19974 ppm, 125 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.6-1.2 (brs, 2.8H), 1.4-2.4 (brm, 7.2H),2.8-3.1 (brm, 2.4H), 3.8-4.5 (brm, 3.8H), 4.5-5.5 (brm, 0.3H), 6.4-6.6(brm, 0.2H), 6.7-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 1.7H).

Example 53 Preparation of γ-PGA-PAE (12% K Salt)

γ-PGA-PAE (200 mg, PAE introduction rate: 55%) and DMSO (1.5 mL) weremeasured and stirred at room temperature for 1 hour to acquire asolution. This solution (1.5 mL) was added dropwise into distilled water(28.5 mL). A 0.1 M KOH aqueous solution (2.2 mL) was added dropwise withstirring at room temperature to adjust pH to 6.0. Subsequently, 0.01 MHCl (0.02 mL) was added with stirring at room temperature. This solutionwas frozen and then subjected to lyophilization to acquire γ-PGA-PAE(12% K salt) (232 mg, PAE introduction rate: 47%, K: 33000 ppm, Cl:11743 ppm, 138 kDa).

¹H NMR (500 MHz, DMSO-d₆) δ 0.6-1.2 (brs, 2.9H), 1.4-2.4 (brm, 7.2H),2.8-3.1 (brm, 2.4H), 3.8-4.5 (brm, 4.0H), 4.5-5.5 (brm, 0.4H), 6.4-6.6(brm, 0.3H), 6.7-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.1H).

Example 54 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (28%, Na salt) (2.0 mg, PAE introduction rate: 58%, Example47) and DMSO (0.2 mL) were measured and dissolved with stirring for 2hours at room temperature to acquire a DMSO aqueous solution(concentration: 10 mg/mL) of γ-PGA-PAE (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.50 M NaCl, 30 nm (PDI 0.29); 0.80 MNaCl, 167 nm (PDI 0.21); 0.95 M NaCl, 348 nm (PDI 0.27); 1.10 M NaCl,775 nm (PDI 0.24); 1.25 M NaCl, 898 nm (PDI 0.47); 1.40 M NaCl, 988 nm(PDI 0.37); and 1.55 M NaCl, 994 nm (PDI 0.38).

Example 55 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (28%, Na salt) (2.0 mg, PAE introduction rate: 58%, Example47), DMSO (0.16 mL), and distilled water (0.04 mL) were measured anddissolved with stirring for 2 hours at room temperature to acquire aDMSO aqueous solution (concentration: 10 mg/mL) of γ-PGA-PAE (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.80 M NaCl, 98 nm (PDI 0.21); 0.95 MNaCl, 197 nm (PDI 0.25); 1.10 M NaCl, 357 nm (PDI 0.43); 1.25 M NaCl,577 nm (PDI 0.45); 1.40 M NaCl, 741 nm (PDI 0.51); and 1.55 M NaCl, 649nm (PDI 0.37).

Example 56 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (26%, Na salt) (2.0 mg, PAE introduction rate: 56%, Example48) and DMSO (0.2 mL) were measured and dissolved with stirring for 2hours at room temperature to acquire a DMSO aqueous solution(concentration: 10 mg/mL) of γ-PGA-PAE (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.50 M NaCl, 43 nm (PDI 0.19); 0.65 MNaCl, 145 nm (PDI 0.16); 0.70 M NaCl, 278 M (PDI 0.29); 0.75 M NaCl, 282nm (PDI 0.28); 0.80 M NaCl, 578 nm (PDI 0.43); 0.95 M NaCl, 963 nm (PDI0.27); and 1.10 M NaCl, 1539 nm (PDI 0.26).

Example 57 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (26%, Na salt) (2.0 mg, PAE introduction rate: 56%, Example48), DMSO (0.16 mL), and distilled water (0.04 mL) were measured anddissolved with stirring for 2 hours at room temperature to acquire aDMSO aqueous solution (concentration: 10 mg/mL) of γ-PGA-PAE (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.70 M NaCl, 92 nm (PDI 0.18); 0.75 MNaCl, 135 nm (PDI 0.27); 0.80 M NaCl, 167 nm (PDI 0.19); 0.95 M NaCl,398 nm (PDI 0.28); and 1.10 M NaCl, 677 nm (PDI 0.38).

Example 58 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (14%, Na salt) (2.0 mg, PAE introduction rate: 53%, Example49) and DMSO (0.2 mL) were measured and dissolved with stirring for 2hours at room temperature to acquire a DMSO aqueous solution(concentration: 10 mg/mL) of γ-PGA-PAE (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.40 M NaCl, 77 nm (PDI 0.15); 0.50 MNaCl, 158 nm (PDI 0.11); 0.60 M NaCl, 244 nm (PDI 0.19); 0.65 M NaCl,310 nm (PDI 0.22); and 0.70 M NaCl, 489 nm (PDI 0.25).

Example 59 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (14.%, Na salt) (2.0 mg, PAE introduction rate: 53%, Example49), DMSO (0.16 mL), and distilled water (0.04 mL) were measured anddissolved with stirring for 2 hours at room temperature to acquire aDMSO aqueous solution (concentration: 10 mg/mL) of γ-PGA-PAE (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.60 M NaCl, 113 nm (PDI 0.11); 0.70 MNaCl, 184 nm (PDI 0.20); 0.75 M NaCl, 210 nm (PDI 0.19); 0.80 M NaCl,312 nm (PDI 0.28); 0.90 M NaCl, 553 nm (PDI 0.31); and 1.00 M NaCl, 800nm (PDI 0.27).

Example 60 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (0%, Na salt) (2.0 mg, PAE introduction rate: 51%, Example 50)and DMSO (0.2 mL) were measured and dissolved with stirring for 2 hoursat room temperature to acquire a DMSO aqueous solution (concentration:10 mg/mL) of γ-PGA-PAE (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.00 M NaCl, 155 nm (PDI 0.11); and0.01 M NaCl, 217 nm (PDI 0.09).

Example 61 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (0%, Na salt) (2.0 mg, PAE introduction rate: 51%, Example50), DMSO (0.16 mL), and distilled water (0.04 mL) were measured andadded and dissolved with stirring for 2 hours at room temperature toacquire a DMSO aqueous solution (concentration: 10 mg/mL) of γ-PGA-PAE(Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.00 M NaCl, 140 nm (PDI 0.14); 0.01 MNaCl, 178 nm (PDI 0.07); and 0.02 M NaCl, 220 nm (PDI 0.09).

Example 62 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of Na₂CO₃ (12.0 mg) was added dropwise and stirred for2 hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-PAE (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.95 M NaCl, 192 nm (PDI 0.42); 1.10 MNaCl, 274 nm (PDI 0.28); 1.25 M NaCl, 494 nm (PDI 0.32); 1.40 M NaCl,810 nm (PDI 0.42); and 1.55 M NaCl, 871 nm (PDI 0.42).

Example 63 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of Na₂CO₃ (6.0 mg) was added dropwise and stirred for2 hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-PAE (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 1.00 M NaCl, 184 nm (PDI 0.23); 1.10 MNaCl, 335 nm (PDI 0.27); 1.15 M NaCl, 553 nm (PDI 0.51); 1.20 M NaCl,505 nm (PDI 0.32); 1.25 M NaCl, 619 nm (PDI 0.39); and 1.30 M NaCl, 727nm (PDI 0.39).

Example 64 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of Na₂CO₃ (3.0 mg) was added dropwise and stirred for2 hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-PAE (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.60 M NaCl, 53 nm (PDI 0.20); 0.80 MNaCl, 202 nm (PDI 0.22); 0.85 M NaCl, 245 nm (PDI 0.25); 0.90 M NaCl,355 nm (PDI 0.29); 0.95 M NaCl, 875 nm (PDI 0.59); and 1.00 M NaCl, 676nm (PDI 0.50).

Example 65 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of Na₂CO₃ (1.5 mg) was added dropwise and stirred for2 hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-PAE (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.20 M NaCl, 63 nm (PDI 0.06); 0.25 MNaCl, 78 nm (PDI 0.05); 0.30 M NaCl, 112 nm (PDI 0.08); 0.35 M NaCl, 123nm (PDI 0.09); 0.40 M NaCl, 164 nm (PDI 0.10); 0.45 M NaCl, 210 nm (PDI0.16); 0.50 M NaCl, 459 nm (PDI 0.42); and 0.60 M NaCl, 719 nm (PDI0.36).

Example 66 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of Na₂HPO₄ (12.0 mg) was added dropwise and stirredfor 2 hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL)of γ-PGA-PAE (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.50 M NaCl, 34 nm (PDI 0.21); 0.60 MNaCl, 118 nm (PDI 0.17); 0.70 M NaCl, 197 nm (PDI 0.22); 0.80 M NaCl,345 nm (PDI 0.28); 0.90 M NaCl, 537 nm (PDI 0.35); and 1.00 M NaCl, 642nm (PDI 0.28).

Example 67 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of Na₂HPO₄ (6.0 mg) was added dropwise and stirred for2 hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-PAE (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.05 M NaCl, 31 nm (PDI 0.14); 0.20 MNaCl, 56 nm (PDI 0.07); 0.30 M NaCl, 88 nm (PDI 0.10); 0.40 M NaCl, 137nm (PDI 0.11); 0.50 M NaCl, 236 nm (PDI 0.20); 0.60 M NaCl, 530 nm (PDI0.48); and 0.70 M NaCl, 585 nm (PDI 0.49).

Example 68 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of Na₂HPO₄ (3.0 mg) was added dropwise and stirred for2 hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-PAE (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.01 M NaCl, 365 nm (PDI 0.21); 0.02 MNaCl, 595 nm (PDI 0.21); 0.04 M NaCl, 1195 nm (PDI 0.25); and 0.05 MNaCl, 1057 nm (PDI 0.46).

Example 69 Preparation of Nanoparticles of γ-PGA-PAE (K Salt)

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of K₂CO₃ (12.0 mg) was added dropwise and stirred for2 hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-PAE (K salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into KCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nana ZS (Malvern). When the KCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 1.40 M KCl, 415 nm (PDI 0.55); 1.50 MKCl, 595 nm (PDI 0.41); and 1.60 M KCl, 902 nm (PDI 0.54).

Example 70 Preparation of Nanoparticles of γ-PGA-PAE (K Salt)

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of K₂CO₃ (6.0 mg) was added dropwise and stirred for 2hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-PAE (K salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into KCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the KCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.60 M KCl, 83 nm (PDI 0.29); 0.70 MKCl, 189 nm (PDI 0.25); 0.80 M KCl, 421 nm (PDI 0.30); 0.90 M KCl, 940nm (PDI 0.26); and 0.95 M KCl, 1185 nm (PDI 0.07).

Example 71 Preparation of Nanoparticles of γ-PGA-PAE (K Salt)

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of K₂CO₃ (3.0 mg) was added dropwise and stirred for 2hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-PAE (K salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into KCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the KCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.50 M KCl, 103 nm (PDI 0.11); 0.55 MKCl, 93 nm (PDI 0.12); 0.60 M KCl, 161 nm (PDI 0.16); 0.65 M KCl, 216 nm(PDI 0.22); 0.70 M KCl, 308 nm (PDI 0.23); 0.75 M KCl, 398 nm (PDI0.31); and 0.80 M KCl, 612 nm (PDI 0.31).

Example 72 Preparation of Nanoparticles of γ-PGA-PAE (K Salt)

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of K₂CO₃ (1.5 mg) was added dropwise and stirred for 2hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-PAE (K salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into KCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the KCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.05 M KCl, 105 nm (PDI 0.08); 0.08 MKCl, 153 nm (PDI 0.08); 0.10 M KCl, 198 nm (PDI 0.15); 0.12 M KCl, 266nm (PDI 0.20); 0.15 M KCl, 356 nm (PDI 0.18); and 0.20 M KCl, 838 nm(PDI 0.29).

Example 73 Preparation of Nanoparticles of γ-PGA-PAE (K Salt)

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of KHCO₃ (12.0 mg) was added dropwise and stirred for2 hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-PAE (K salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into KCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the KCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.70 M KCl, 127 nm (PDI 0.20); 0.80 MKCl, 263 nm (PDI 0.28); 0.90 M KCl, 479 nm (PDI 0.42); 1.00 M KCl, 1097nm (PDI 0.47); and 1.10 M KCl, 2085 nm (PDI 0.15).

Example 74 Preparation of Nanoparticles of γ-PGA-PAE (K Salt)

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of KHCO₃ (6.0 mg) was added dropwise and stirred for 2hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-PAE (K salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into KCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the KCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.50 M KCl, 40 nm (PDI 0.16); 0.60 MKCl, 63 nm (PDI 0.18); 0.70 M KCl, 141 nm (PDI 0.18); 0.80 M KCl, 286 nm(PDI 0.27); 0.90 M KCl, 679 nm (PDI 0.49); and 1.00 M KCl, 1228 nm (PDI0.04).

Example 75 Preparation of Nanoparticles of γ-PGA-PAE (K Salt)

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of KHCO₃ (3.0 mg) was added dropwise and stirred for 2hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-PAE (K salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into KCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the KCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.20 M KCl, 58 nm (PDI 0.07); 0.30 MKCl, 91 nm (PDI 0.07); 0.40 M KCl, 161 nm (PDI 0.11); 0.45 M KCl, 189 nm(PDI 0.15); 0.50 M KCl, 303 nm (PDI 0.25); 0.55 M KCl, 488 nm (PDI0.31); and 0.60 M KCl, 672 nm (PDI 0.28).

Example 76 Preparation of Nanoparticles of γ-PGA-PAE (K Salt)

γ-PGA-PAE (30 mg, PAE introduction rate: 55%) and DMSO (2.4 mL) weremeasured and stirred at room temperature. Subsequently, an aqueoussolution (0.6 mL) of KHCO₃ (1.5 mg) was added dropwise and stirred for 2hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-PAE (K salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into KCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-PAE were measured in terms ofmean particle diameter [Z-Ave d, (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the KCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.00 M KCl, 237 nm (PDI 0.11); 0.005 MKCl, 242 nm (PDI 0.11); and 0.01 M KCl, 361 nm (PDI 0.17).

Example 77 Preparation of Nanoparticles of γ-PGA-PAE

In a 100-mL egg-plant shaped flask, γ-PGA-PAE (1.03 g, moisture: 2.21%)and DMSO (60 mL) were measured, stirred at room temperature, andcompletely dissolved. At an internal temperature of 18 to 30° C., NaHCO₃aqueous solution [NaHCO₃ (50 mg) was diluted with distilled water (20.0mL)] was added dropwise. After stirring for 30 minutes, an acquiredsolution was filtered with a syringe filter (Corning, 0.2 μm). Afterwashing with DMSO wash liquid (20 mL), the wash liquid waswashed/filtered through a syringe filter and mixed. This solution wasdefined as a solution A (0.5 mL was sampled for study).

NaCl (2.63 g) was dissolved in Otsuka distilled water (100 mL) andfiltered with a syringe filter (0.2 μm). This solution was defined as asolution B.

The solutions A and B were mixed through a ⅛″ tube at a flow rate of 10mL/minute. The mixed solution was received by a 1000-mL plastic bottle(Corning, storage bottle made of PS, 430281). Distilled water (about 800mL) was added to this mixed solution to prepare a solution fordesalting. This solution was desalted (desalting conditions: SARTOCON(registered trademark) Slice Cassette 2 kDa, TM 20-22 psi). Thissolution was frozen in a freezer at −40° C. This frozen solution wassubjected to lyophilization to acquire nanoparticles of γ-PGA-PAE [957mg, yield: 95.7%, moisture: 1.40%, PAE introduction rate: 57%, Z-Ave d.522 nm (PDI 0.49)].

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.2H), 1.4-2.4 (brm, 7.1H),2.6-2.8 (brm, 0.2H), 2.8-3.1 (brm, 2.3H), 3.9-4.5 (brm, 4.8H), 4.5-5.5(brm, 0.3H), 6.9-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.5H).

Example 78 Preparation of Nanoparticles of γ-PGA-PAE

In a 100-mL egg-plant shaped flask, γ-PGA-PAE (3.075 g, moisture: 2.21%)and DMSO (45 mL) were measured, stirred at room temperature, andcompletely dissolved. At an internal temperature of 18 to 30° C., NaHCO₃aqueous solution [acquired by diluting NaHCO₃ (300 mg) with distilledwater (6.0 mL), washing with distilled water (3.0 mL) after dropping,and further dropping this wash liquid] was added dropwise. Afterstirring for 30 minutes, an acquired solution was filtered with asyringe filter (Corning, 0.2 μm). After washing with DMSO wash liquid (6mL), the wash liquid was washed/filtered through a syringe filter andmixed. This solution was defined as a solution A (0.5 mL was sampled forstudy).

In a 1-L plastic bottle (Corning, storage bottle made of PS, 430281),NaCl (23.38 g) and Otsuka distilled water (1000 mL) were measured anddissolved. This solution was filtered by a 0.2 μm filter. This solutionwas defined as a solution B.

The solution A (about 60 mL) and the solution B (about 70 mL) were mixedthrough a ⅛″ tube at a flow rate of 10 mL/minute. The mixed solution wasreceived by a 1-L plastic bottle (Corning, storage bottle made of PS,430281). Distilled water (about 900 mL) was added to this mixed solutionto prepare a solution for desalting. This solution was desalted[desalting conditions: SARTOCON (registered trademark) Slice Cassette 10kDa (3051443901E-SG), TM 17-18 psi, about 12 to 16 g/minute, about 3hours]. This solution was frozen in a freezer at −40° C. This frozensolution was subjected to lyophilization to acquire nanoparticles ofγ-PGA-PAE [2.68 g, yield: 83.4%, moisture: 6.72%, PAE introduction rate:57%, Z-Ave d. 79.5 nm (PDI 0.28)].

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.2H), 1.4-2.4 (brm, 7.0H),2.8-3.1 (brm, 2.3H), 3.9-4.5 (brm, 4.5H), 4.5-5.5 (brm, 0.2H), 6.9-7.5(brm, 5.0H, a relative value when the protons of the phenyl group of thephenylalanyl group are assumed as 5.0H), 7.6-8.7 (brm, 2.5H). 1H NMR(500 MHz, D₂O) 5 No signal. When a gain was increased for forcibleobservation, the following extremely small peaks were observed: 0.7-1.2(brs, 3.0H), 1.5-2.5 (brm, 5.1H), 2.7-2.3 (brm, 1.8H), 3.7-4.2 (brm,2.0H), 6.7-7.4 (brm, 5.0H, a relative value when the protons of thephenyl group of the phenylalanyl group are assumed as 5.0H).

Example 79 Preparation of Nanoparticles of γ-PGA-PAE

In a 100-mL egg-plant shaped flask, γ-PGA-PAE (3.075 g, moisture: 2.21%)and DMSO (45 mL) were measured, stirred at room temperature, andcompletely dissolved. At an internal temperature of 18 to 30° C., NaOHaqueous solution [acquired by diluting 1 M NaOH (2.5 mL) with distilledwater (4.5 mL), washing with distilled water (2.0 mL) after dropping,and further dropping this wash liquid] was added dropwise. Afterstirring for 30 minutes, an acquired solution was filtered with asyringe filter (Corning, 0.2 μm). After washing with DMSO wash liquid (6mL), the wash liquid was washed/filtered through a syringe filter andmixed. This solution was defined as a solution A.

In a 1-L plastic bottle (Corning, storage bottle made of PS, 430281),NaCl (17.53 g) and Otsuka distilled water (1000 mL) were measured anddissolved. This solution was filtered by a 0.2 μm filter. This solutionwas defined as a solution B (0.5 mL was sampled for study).

The solution A (about 60 mL) and the solution B (about 70 mL) were mixedthrough a ⅛″ tube at a flow rate of 10 mL/minute. The mixed solution wasreceived by a 1-L Plastic bottle (Corning, storage bottle made of PS,430281). Distilled water (about 900 mL) was added to this mixed solutionto prepare a solution for desalting. This solution was desalted[desalting conditions: SARTOCON (registered trademark) Slice Cassette 10kDa (3051443901E-SG), TM 17-18 psi, about 12 to 16 g/minute, about 3hours]. This solution was frozen in a freezer at −40° C. This frozensolution was subjected to lyophilization to acquire nanoparticles ofγ-PGA-PAE [2.76 g, yield: 88.7%, moisture: 3.50%, PAE introduction rate:57%, Z-Ave d. 80.2 nm (PDI 0.21)].

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.2H), 1.4-2.4 (brm, 7.1H),2.6-2.8 (brm, 0.2H), 2.8-3.1 (brm, 2.3H), 3.9-4.5 (brm, 4.9H), 4.5-5.5(brm, 0.3H), 6.9-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.7H).

Example 80 Preparation of Nanoparticles of γ-PGA-PAE

In a 100-mL egg-plant shaped flask, γ-PGA-PAE (3.075 g, moisture: 2.21%)and DMSO (45 mL) were measured, stirred at room temperature, andcompletely dissolved. At an internal temperature of 18 to 30° C., Na₂CO₃aqueous solution [acquired by diluting Na₂CO₃ (189 mg) with distilledwater (6.0 mL), washing with distilled water (3.0 mL) after dropping,and further dropping this wash liquid] was added dropwise. Afterstirring for 30 minutes, an acquired solution was filtered with asyringe filter (Corning, 0.2 μm). After washing with DMSO wash liquid (6mL), the wash liquid was washed/filtered through a syringe filter andmixed. This solution was defined as a solution A (0.5 mL was sampled forstudy).

In a 1-L plastic bottle (Corning, storage bottle made of PS, 430281),NaCl (25.13 g) and Otsuka distilled water (1000 mL) were measured anddissolved. This solution was filtered by a 0.2 μm filter. This solutionwas defined as a solution B.

The solution A (about 60 mL) and the solution B (about 70 mL) were mixedthrough a ⅛″ tube at a flow rate of 10 mL/minute. The mixed solution wasreceived by a 1-L plastic bottle (Corning, storage bottle made of PS,430281). Distilled water (about 900 mL) was added to this mixed solutionto prepare a solution for desalting. This solution was desalted[desalting conditions: SARTOCON (registered trademark) Slice Cassette 10kDa (3051443901E-SG), TM 17-18 psi, about 12 to 16 g/minute, about 3hours]. This solution was frozen in a freezer at −40° C. This frozensolution was subjected to lyophilization to acquire nanoparticles ofγ-PGA-PAE [2.75 g, yield: 87.3%, moisture: 4.64%, PAE introduction rate:56%, Z-Ave d. 98.5 nm (PDI 0.25)].

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.3H), 1.4-2.4 (brm, 7.1H),2.6-2.8 (brm, 0.2H), 2.8-3.1 (brm, 2.3H), 3.9-4.5 (brm, 4.7H), 4.5-5.5(brm, 0.2H), 6.9-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.5H).

Example 81 Preparation of Nanoparticles of γ-PGA-PAE

In a 100-mL egg-plant shaped flask, γ-PGA-PAE (3.075 g, moisture: 2.21%)and DMSO (45 mL) were measured, stirred at room temperature, andcompletely dissolved. At an internal temperature of 18 to 30° C., Na₂CO₃aqueous solution [acquired by diluting Na₂CO₃ (189 mg) with distilledwater (6.0 mL), washing with distilled water (3.0 mL) after dropping,and further dropping this wash liquid] was added dropwise. Afterstirring for 30 minutes, an acquired solution was filtered with asyringe filter (Corning, 0.2 μm). After washing with DMSO wash liquid (6mL), the wash liquid was washed/filtered through a syringe filter andmixed. This solution was defined as a solution A (0.5 mL was sampled forstudy).

In a 1-L plastic bottle (Corning, storage bottle made of PS, 430281),NaCl (17.53 g) and Otsuka distilled water (1000 mL) were measured anddissolved. This solution was filtered by a 0.2 μm filter. This solutionwas defined as a solution B.

The solution A (about 60 mL) and the solution B (about 70 mL) were mixedthrough a ⅛″ tube at a flow rate of 10 mL/minute. The mixed solution wasreceived by a 1-L plastic bottle (Corning, storage bottle made of PS,430281). Distilled water (about 900 mL) was added to this mixed solutionto prepare a solution for desalting. This solution was desalted[desalting conditions: SARTOCON (registered trademark) Slice Cassette 10kDa (3051443901E-SG), TM 17-18 psi, about 12 to 16 g/minute, about 3hours]. This solution was frozen in a freezer at −40° C. This frozensolution was subjected to lyophilization to acquire nanoparticles ofγ-PGA-PAE [2.63 g, yield: 83.4%, moisture: 4.86%, PAE introduction rate:57%, Z-Ave d. 261.5 nm (PDI 0.40)].

¹H NMR (500 MHz, DMSO-d₆) δ 0.9-1.3 (brs, 3.2H), 1.4-2.4 (brm, 7.0H),2.6-2.8 (brm, 0.2H), 2.8-3.1 (brm, 2.2H), 3.9-4.5 (brm, 4.7H), 4.5-5.5(brm, 0.2H), 6.9-7.5 (brm, 5.0H, a relative value when the protons ofthe phenyl group of the phenylalanyl group are assumed as 5.0H), 7.6-8.7(brm, 2.4H).

Example 82 Preparation of Nanoparticles of γ-PGA-PAE

In a 100-mL egg-plant shaped flask, γ-PGA-PAE (3.075 g, moisture: 2.21%)and DMSO (45 mL) were measured, stirred at room temperature, andcompletely dissolved. At an internal temperature of 18 to 30° C., Na₂CO₃aqueous solution [acquired by diluting Na₂CO₃ (189 mg) with distilledwater (6.0 mL), washing with distilled water (3.0 mL) after dropping,and further dropping this wash liquid] was added dropwise. Afterstirring for 30 minutes, an acquired solution was filtered with asyringe filter (Corning, 0.2 μm). After washing with DMSO wash liquid (6mL), the wash liquid was washed/filtered through a syringe filter andmixed. This solution was defined as a solution A (0.5 mL of thissolution was sampled and used for this study).

A portion of this solution A (25 μL) was quickly mixed into PBS aqueoussolutions (25 μL) of various concentrations to acquire dispersionliquid. This dispersion liquid was used for measuring the nanoparticlesof γ-PGA-PAE in terms of mean particle diameter [Z-Ave d. (nm)] andparticle diameter dispersion index (PDI) by Zetasizer Nano ZS (Malvern).When the PBS aqueous solutions of various concentrations were used forpreparation, the following results were acquired: ×1 PBS, 44 nm (PDI0.24); ×2 PBS, 43 nm (PDI 0.23); ×3 PBS, 74 nm (PDI 0.23); ×3.1 PBS, 89nm (PDI 0.25); and ×3.2 PBS, 138 nm (PDI 0.43).

Example 83 Preparation of Nanoparticles of γ-PGA-PAE

In a 100-mL egg-plant shaped flask, γ-PGA-PAE (3.075 g, moisture: 2.21%)and DMSO (45 mL) were measured, stirred at room temperature, andcompletely dissolved. At an internal temperature of 18 to 30° C., NaHCO₃aqueous solution [acquired by diluting NaHCO₃ (300 mg) with distilledwater (6.0 mL), washing with distilled water (3.0 mL) after dropping,and further dropping this wash liquid] was added dropwise. Afterstirring for 30 minutes, an acquired solution was filtered with asyringe filter (Corning, 0.2 μm). After washing with DMSO wash liquid (6mL), the wash liquid was washed/filtered through a syringe filter andmixed. This solution was defined as a solution A (0.5 mL of thissolution was sampled and used for this study).

A portion of this solution A (25 μL) was quickly mixed into AcONaaqueous solutions (25 μL) of various concentrations to acquiredispersion liquid. This dispersion liquid was used for measuring thenanoparticles of γ-PGA-PAE in terms of mean particle diameter [Z-Ave d.(nm)] and particle diameter dispersion index (PDI) by Zetasizer Nano ZS(Malvern). When the AcONa aqueous solutions of various concentrationswere used for preparation, the following results were acquired: 0.1 MAcONa, 38 nm (PDI 0.25); 0.2 M AcONa, 36 nm (PDI 0.23); 0.3 M AcONa, 39nm (PDI 0.23); and 0.4 M AcONa, 68 nm (PDI 0.38).

Example 84 Preparation of Nanoparticles of γ-PGA-PAE

In a 5-mL sample bottle, γ-PGA-PAE (60 mg, moisture: 2.21%) and EtOH (4mL) were measured, stirred at room temperature, and suspended. At roomtemperature, NaHCO₃ aqueous solution [Na₂CO₃ (63 mg) was diluted withdistilled water (3 mL) and 0.36 mL of this solution was used] was addeddropwise. After stirring for 30 minutes, distilled water (1 mL) wasadded dropwise and an acquired solution was filtered with a syringefilter (Corning, 0.2 μm). This solution was defined as a solution A.

A portion of this solution A (50 μL) was quickly mixed into NaCl aqueoussolutions (50 μL) of various concentrations to acquire dispersionliquid. This dispersion liquid was used for measuring the nanoparticlesof γ-PGA-PAE in terms of mean particle diameter [Z-Ave d. (nm)] andparticle diameter dispersion index (PDI) by Zetasizer Nano ZS (Malvern).When the NaCl aqueous solutions of various concentrations were used forpreparation, the following results were acquired: 0.3 M NaCl, 35 nm (PDI0.39); 0.4 M NaCl, 63 nm (PDI 0.48); 0.45 M NaCl, 206 nm (PDI 0.46); and0.5 M NaCl, 415 nm (PDI 0.35).

Example 85 Preparation of Nanoparticles of γ-PGA-Phe-OcPen

γ-PGA-Phe-OcPen (30 mg, Phe-OcPen introduction rate: 57%) and DMSO (2.4mL) were measured and stirred at room temperature. NaOH aqueous solution(0.6 mL, equivalent to 0.051 mmol) was then added dropwise. The solutionwas stirred for 2 hours to acquire a DMSO aqueous solution(concentration: 10 mg/mL) of γ-PGA-Phe-OcPen (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-Phe-OcPen (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-Phe-OcPen were measured interms of mean particle diameter [Z-Ave d. (nm)] and particle diameterdispersion index (PDI) by Zetasizer Nano ZS (Malvern). When the NaClaqueous solutions of various concentrations were used for preparation,the following results were acquired: 0.40 M NaCl, 39 nm (PDI 0.19); 0.50M NaCl, 70 nm (PDI 0.18); 0.60 M NaCl, 124 nm (PDI 0.16); 0.70 M NaCl,199 nm (PDI 0.25); 0.80 M NaCl, 314 nm (PDI 0.28); and 0.90 M NaCl, 670nm (PDI 0.59).

Example 86 Preparation of Nanoparticles of γ-PGA-Phe-OtBu

γ-PGA-Phe-OtBu (30 mg, Phe-OtBu introduction rate: 57%) and DMSO (2.4mL) were measured and stirred at room temperature. An aqueous solution(0.6 mL) of NaHCO₃ (3.0 mg) was then added dropwise and stirred for 2hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-Phe-OtBu (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-Phe-OtBu (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-Phe-OtBu were measured in termsof mean particle diameter [Z-Ave d. (nm)] and particle diameterdispersion index (PDI) by Zetasizer Nano ZS (Malvern). When the NaClaqueous solutions of various concentrations were used, the followingresults were acquired: 0.20 M NaCl, 45 nm (PDI 0.25); 0.30 M NaCl, 97 nm(PDI 0.16); 0.40 M NaCl, 311 nm (PDI 0.27); 0.45 M NaCl, 685 nm (PDI0.22); and 0.50 M NaCl, 1066 nm (PDI 0.37).

Example 87 Preparation of Nanoparticles of γ-PGA-Phe-OtBu

γ-PGA-Phe-OtBu (30 mg, Phe-OtBu introduction rate: 57%) and DMSO (2.4mL) were measured and stirred at room temperature. An aqueous solution(0.6 mL) of NaHCO₃ (1.5 mg) was then added dropwise and stirred for 2hours to acquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-Phe-OtBu (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-Phe-OtBu (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-Phe-OtBu were measured in termsof mean particle diameter [Z-Ave d. (nm)] and particle diameterdispersion index (PDI) by Zetasizer Nano ZS (Malvern). When the NaClaqueous solutions of various concentrations were used for preparation,the following results were acquired: 0.20 M NaCl, 49 nm (PDI 0.46); 0.40M NaCl, 80 nm (PDI 0.29); 0.50 M NaCl, 123 nm (PDI 0.28); 0.60 M NaCl,202 nm (PDI 0.41); and 0.70 M NaCl, 414 nm (PDI 0.45).

Example 88 Preparation of Nanoparticles of γ-PGA-Phe-OtBu

γ-PGA-Phe-OtBu (0.90 g, Phe-OtBu introduction rate: 57%) and DMSO (7.2mL) were measured and stirred at room temperature. NaOH aqueous solution(1.8 mL, equivalent to 1.6 mmol) was added dropwise. The solution wasstirred for 2 hours to acquire a DMSO aqueous solution (concentration:100 mg/mL) of γ-PGA-Phe-OtBu (Na salt).

First, preliminary examination was conducted with NaCl aqueous solution.A portion of the DMSO aqueous solution (concentration: 100 mg/mL, 0.1mL) of γ-PGA-Phe-OtBu (Na salt) was quickly mixed into NaCl aqueoussolutions (0.1 mL) of various concentrations to acquire dispersionliquid. The liquid was desalted by using Amicon Ultra 0.5 (Millipore, 10K) (centrifugation conditions: 4500 rpm, 30 minutes, 5° C.). Anoperation of dispersing an acquired residue in distilled water (0.1 mL)followed by desalting was performed twice. The acquired residue wasdispersed in ×1 PBS (1.0 mL) to acquire a solution of nanoparticles ofγ-PGA-Phe-OtBu (10 mg/mL). The nanoparticles were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used, the following resultswere acquired: 0.20 M NaCl, 47 nm (PDI 0.24); 0.45 M NaCl, 64 nm (PDI0.22); 0.50 M NaCl, 80 nm (PDI 0.23); and 0.55 M NaCl, 140 nm (PDI0.42).

The scale was then increased. The DMSO aqueous solution (concentration:100 mg/mL, 8.0 mL) of γ-PGA-Phe-OtBu (Na salt) described above wasquickly mixed into 0.60 M NaCl aqueous solution (8.0 mL) to acquiredispersion liquid. The dispersion liquid was desalted and washed withwater through centrifugal filtration (centrifugation conditions: 4500rpm, 30 minutes, 5° C.). The acquired residue was dispersed in distilledwater (25 mL) and subjected to lyophilization to acquire nanoparticles(849 mg) of γ-PGA-Phe-OtBu. The nanoparticles were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). The result was 105 nm (PDI0.40).

Example 89 Preparation of Nanoparticles of γ-PGA-Phe-OMe

γ-PGA-Phe-OMe (30 mg, Phe-OMe introduction rate: 53%) and DMSO (2.4 mL)were measured and stirred at room temperature. An aqueous solution (0.6mL) of NaHCO₃ (3.0 mg) was added dropwise and stirred for 2 hours toacquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-Phe-OMe (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-Phe-OMe (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-Phe-OMe were measured in termsof mean particle diameter [Z-Ave d. (nm)] and particle diameterdispersion index (PDI) by Zetasizer Nano ZS (Malvern). When the NaClaqueous solutions of various concentrations were used for preparation,the following results were acquired: 0.20 M NaCl, 76 nm (PDI 0.11); 0.30M NaCl, 154 nm (PDI 0.12); 0.35 M NaCl, 196 nm (PDI 0.14); 0.40 M NaCl,273 nm (PDI 0.20); and 0.50 M NaCl, 468 nm (PDI 0.30).

Example 90 Preparation of Nanoparticles of γ-PGA-Phe-OMe

γ-PGA-Phe-OMe (30 mg, Phe-OMe introduction rate: 53%) and DMSO (2.4 mL)were measured and stirred at room temperature. An aqueous solution (0.6mL) of NaHCO₃ (1.5 mg) was added dropwise and stirred for 2 hours toacquire a DMSO aqueous solution (concentration: 10 mg/mL) ofγ-PGA-Phe-OMe (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-Phe-OMe (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-Phe-OMe were measured in termsof mean particle diameter [Z-Ave d. (nm)] and particle diameterdispersion index (PDI) by Zetasizer Nano ZS (Malvern). When the NaClaqueous solutions of various concentrations were used for preparation,the following results were acquired: 0.15 M NaCl, 114 nm (PDI 0.05);0.20 M NaCl, 173 nm (PDI 0.08); 0.25 M NaCl, 333 nm (PDI 0.26); 0.30 MNaCl, 778 nm (PDI 0.42); and 0.40 M NaCl, 2673 nm (PDI 0.17).

Example 91 Preparation of Nanoparticles of γ-PGA-Phe-OMe

γ-PGA-Phe-OMe (0.90 g, Phe-OMe introduction rate: 53%) and DMSO (7.2 mL)were measured and stirred at room temperature. NaOH aqueous solution(1.8 mL, equivalent to 2.0 mmol) was then added dropwise. The solutionwas stirred for 2 hours to acquire a DMSO aqueous solution(concentration: 100 mg/mL) of γ-PGA-Phe-OMe (Na salt).

First, preliminary examination was conducted with NaCl aqueous solution.A portion of the DMSO aqueous solution (concentration: 100 mg/mL, 0.1mL) of γ-PGA-Phe-OMe (Na salt) was quickly mixed into NaCl aqueoussolutions (0.1 mL) of various concentrations to acquire dispersionliquid. The liquid was desalted by using Amicon Ultra 0.5 (Millipore, 10K) (centrifugation conditions: 4500 rpm, 30 minutes, 5° C.). Anoperation of dispersing an acquired residue in distilled water (0.1 mL)followed by desalting was performed twice. The acquired residue wasdispersed in ×1 PBS (1.0 mL) to acquire a solution of nanoparticles ofγ-PGA-Phe-OMe (10 mg/mL). The nanoparticles were measured in terms ofmean particle diameter [Z-Ave d. (nm)] and particle diameter dispersionindex (PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueoussolutions of various concentrations were used for preparation, thefollowing results were acquired: 0.20 M NaCl, 65 nm (PDI 0.30); 0.45 MNaCl, 77 nm (PDI 0.29); 0.60 M NaCl, 98 nm (PDI 0.39); 0.65 M NaCl, 114nm (PDI 0.44); 0.70 M NaCl, 135 nm (PDI 0.78); and 0.75 M NaCl, 170 nm(PDI 0.94).

The scale was then increased. The DMSO aqueous solution (concentration:100 mg/mL, 8.0 mL) of γ-PGA-Phe-OMe (Na salt) described above wasquickly mixed into 0.70 M NaCl aqueous solution (8.0 mL) to acquiredispersion liquid. The dispersion liquid was repeatedly desalted andwashed with water through centrifugal filtration (centrifugationconditions: 4500 rpm, 30 minutes, 5° C.). The acquired residue wasdispersed in distilled water (25 mL) and subjected to lyophilization toacquire nanoparticles (857 mg) of γ-PGA-Phe-OMe. The nanoparticles weremeasured in terms of mean particle diameter [Z-Ave d. (nm)] and particlediameter dispersion index (PDI) by Zetasizer Nano ZS (Malvern). Theresult was 145 nm (PDI 0.57).

Example 92 Preparation of Nanoparticles of γ-PGA-p-F-Phe-OEt

γ-PGA-p-F-Phe-OEt (30 mg, p-F-Phe-OEt introduction rate: 56%) and DMSO(2.4 mL) were measured and stirred at room temperature. NaOH aqueoussolution (0.6 mL, equivalent to 0.056 mmol) was then added dropwise andstirred for 2 hours to acquire a DMSO aqueous solution (concentration:10 mg/mL) of γ-PGA-p-F-Phe-OEt (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-p-F-Phe-OEt (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-p-F-Phe-OEt were measured interms of mean particle diameter [Z-Ave d. (nm)] and particle diameterdispersion index (PDI) by Zetasizer Nano ZS (Malvern). When the NaClaqueous solutions of various concentrations were used for preparation,the following results were acquired: 0.40 M NaCl, 28 nm (PDI 0.12); 0.50M NaCl, 55 nm (PDI 0.10); 0.70 M NaCl, 107 nm (PDI 0.22); 0.80 M NaCl,244 nm (PDI 0.23); 0.90 M NaCl, 545 nm (PDI 0.30); 0.95 M NaCl, 782 nm(PDI 0.27); and 1.10 M NaCl, 897 nm (PDI 0.25).

Example 93 Preparation of Nanoparticles of γ-PGA-p-Cl-Phe-OEt

γ-PGA-p-Cl-Phe-OEt (0.90 g, p-Cl-Phe-OEt introduction rate: 58%) andDMSO (7.2 mL) were measured and stirred at room temperature. NaOHaqueous solution (1.8 mL, equivalent to 1.5 mmol) was then addeddropwise. The solution was stirred for 2 hours to acquire a DMSO aqueoussolution (concentration: 100 mg/mL) of γ-PGA-p-Cl-Phe-OEt (Na salt).

First, preliminary examination was conducted with NaCl aqueous solution.A portion of the DMSO aqueous solution (concentration: 100 mg/mL, 0.1mL) of γ-PGA-p-Cl-Phe-OEt (Na salt) was quickly mixed into NaCl aqueoussolutions (0.1 mL) of various concentrations to acquire dispersionliquid. The liquid was desalted by using Amicon Ultra 0.5 (Millipore, 10K) (centrifugation conditions: 4500 rpm, 30 minutes, 5° C.). Anoperation of dispersing an acquired residue in distilled water (0.1 mL)followed by desalting was performed twice. The acquired residue wasdispersed in ×1 PBS (1.0 mL) to acquire a solution of nanoparticles ofγ-PGA-p-Cl-Phe-OEt (10 mg/mL). The nanoparticles were measured in termsof mean particle diameter [Z-Ave d. (nm)] and particle diameterdispersion index (PDI) by Zetasizer Nano ZS (Malvern). When the NaClaqueous solutions of various concentrations were used for preparation,the following results were acquired: 0.20 M NaCl, 36 nm (PDI 0.26); 0.25M NaCl, 76 nm (PDI 0.24); and 0.30 M NaCl, 492 nm (PDI 0.40).

The scale was then increased. The DMSO aqueous solution (concentration:100 mg/mL, 8.0 mL) of γ-PGA-p-Cl-Phe-OEt (Na salt) described above wasquickly mixed into 0.27 M NaCl aqueous solution (8.0 mL) to acquiredispersion liquid. The dispersion liquid was repeatedly desalted andwashed with water through centrifugal filtration (centrifugationconditions: 4500 rpm, 30 minutes, 5° C.). The acquired residue wasdispersed in distilled water (25 mL) and subjected to lyophilization toacquire nanoparticles (839 mg) of γ-PGA-p-Cl-Phe-OEt. The nanoparticleswere measured in terms of mean particle diameter [Z-Ave d. (nm)] andparticle diameter dispersion index (PDI) by Zetasizer Nano ZS (Malvern).The result was 67 nm (PDI 0.29).

Example 94 Preparation of Nanoparticles of γ-PGA-p-Br-Phe-OEt

γ-PGA-p-Br-Phe-OEt (0.90 g, p-Br-Phe-OEt introduction rate: 56%) andDMSO (7.2 mL) were measured and stirred at room temperature. NaOHaqueous solution (1.8 mL, equivalent to 1.5 mmol) was then addeddropwise. The solution was stirred for 2 hours to acquire a DMSO aqueoussolution (concentration: 100 mg/mL) of γ-PGA-p-Br-Phe-OEt (Na salt).

First, preliminary examination was conducted with NaCl aqueous solution.A portion of the DMSO aqueous solution (concentration: 100 mg/mL, 0.1mL) of γ-PGA-p-Br-Phe-OEt (Na salt) was quickly mixed into NaCl aqueoussolutions (0.1 mL) of various concentrations to acquire dispersionliquid. The liquid was desalted by using Amicon Ultra 0.5 (Millipore,10K) (centrifugation conditions: 4500 rpm, 30 minutes, 5° C.). Anoperation of dispersing an acquired residue in distilled water (0.1 mL)followed by desalting was performed twice. The acquired residue wasdispersed in ×1 PBS (1.0 mL) to acquire a solution of nanoparticles ofγ-PGA-p-Br-Phe-OEt (10 mg/mL). The nanoparticles were measured in termsof mean particle diameter [Z-Ave d. (nm)] and particle diameterdispersion index (PDI) by Zetasizer Nano ZS (Malvern). When the NaClaqueous solutions of various concentrations were used for preparation,the following results were acquired: 0.30 M NaCl, 73 nm (PDI 0.19); 0.35M NaCl, 131 nm (PDI 0.25); 0.40 M NaCl, 226 nm (PDI 0.27); and 0.45 MNaCl, 508 nm (PDI 0.40).

The scale was then increased. The DMSO aqueous solution (concentration:100 mg/mL, 8.0 mL) of γ-PGA-p-Br-Phe-OEt (Na salt) described above wasquickly mixed into 0.32 M NaCl aqueous solution (8.0 mL) to acquiredispersion liquid. The dispersion liquid was repeatedly desalted andwashed with water through centrifugal filtration (centrifugationconditions: 4500 rpm, 30 minutes, 5° C.). The acquired residue wasdispersed in distilled water (25 mL) and subjected to lyophilization toacquire nanoparticles (632 mg) of γ-PGA-p-Br-Phe-OEt. The nanoparticleswere measured in terms of mean particle diameter [Z-Ave d. (nm)] andparticle diameter dispersion index (PDI) by Zetasizer Nano ZS (Malvern).The result was 52 nm (PDI 0.27).

Example 95 Preparation of Nanoparticles of γ-PGA-p-NO₂-Phe-OEt

γ-PGA-p-NO₂-Phe-OEt (30 mg, p-NO₂-Phe-OEt introduction rate: 58%) andDMSO (2.4 mL) were measured and stirred at room temperature. NaOHaqueous solution (0.6 mL, equivalent to 0.049 mmol) was then addeddropwise and stirred for 2 hours to acquire a DMSO aqueous solution(concentration: 10 mg/mL) of γ-PGA-p-NO₂-Phe-OEt (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-p-NO₂-Phe-OEt (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-p-NO₂-Phe-OEt were measured interms of mean particle diameter [Z-Ave d. (nm)] and particle diameterdispersion index (PDI) by Zetasizer Nano ZS (Malvern). When the NaClaqueous solutions of various concentrations were used for preparation,the following results were acquired: 0.30 M NaCl, 23 nm (PDI 0.32); 0.40M NaCl, 32 nm (PDI 0.37); 0.45 M NaCl, 86 nm (PDI 0.26); and 0.50 MNaCl, 368 nm (PDI 0.31).

Example 96 Preparation of Nanoparticles of γ-PGA-p-OiPr-Phe-OEt

γ-PGA-p-OiPr-Phe-OEt (30 mg, p-OiPr-Phe-OEt introduction rate: 57%) andDMSO (2.4 mL) were measured and stirred at room temperature. NaOHaqueous solution (0.6 mL, equivalent to 0.049 mmol) was then addeddropwise and stirred for 2 hours to acquire a DMSO aqueous solution(concentration: 10 mg/mL) of γ-PGA-p-OiPr-Phe-OEt (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-p-OiPr-Phe-OEt (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-p-OiPr-Phe-OEt were measured interms of mean particle diameter [Z-Ave d. (nm)] and particle diameterdispersion index (PDI) by Zetasizer Nano ZS (Malvern). When the NaClaqueous solutions of various concentrations were used for preparation,the following results were acquired: 0.20 M NaCl, 24 nm (PDI 0.31); 0.25M NaCl, 23 nm (PDI 0.25); 0.30 M NaCl, 36 nm (PDI 0.37); 0.35 M NaCl, 42nm (PDI 0.39); 0.40 M NaCl, 43 nm (PDI 0.29); and 0.50 M NaCl, 44 nm(PDI 0.30).

Example 97 Preparation of Nanoparticles of γ-PGA-α-Phegly-OEt

γ-PGA-α-Phegly-OEt (30 mg, α-Phegly-OEt introduction rate: 59%) and DMSO(2.4 mL) were measured and stirred at room temperature. NaOH aqueoussolution (0.6 mL, equivalent to 0.055 mmol) was then added dropwise andstirred for 2 hours to acquire a DMSO aqueous solution (concentration:10 mg/mL) of γ-PGA-α-Phegly-OEt (Na salt).

A portion of this solution (concentration: 10 mg/mL, 0.2 mL) was quicklymixed into NaCl aqueous solutions (0.2 mL) of various concentrations toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra0.5 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (0.2 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in ×1 PBS (0.2 mL) to acquire a solution ofnanoparticles of γ-PGA-α-Phegly-OEt (10 mg/mL).

Subsequently, the nanoparticles of γ-PGA-α-Phegly-OEt were measured interms of mean particle diameter [Z-Ave d. (nm)] and particle diameterdispersion index (PDI) by Zetasizer Nano ZS (Malvern). When the NaClaqueous solutions of various concentrations were used for preparation,the following results were acquired: 0.40 M NaCl, 32 nm (PDI 0.43); 0.45M NaCl, 124 nm (PDI 0.81); and 0.50 M NaCl, 809 nm (PDI 0.52).

Example 98 Preparation of Nanoparticles of αβ-DL-PolyAsp-D-PA

In a 5-mL sample bottle, αβ-DL-PolyAsp-D-PAE (50 mg, PAE introductionrate: 63%) and DMSO (0.5 mL) were measured and dissolved at roomtemperature. At room temperature, Na₂CO₃ aqueous solution was addeddropwise [0.15 mL of solution acquired by dissolving Na₂CO₃ (63 mg) indistilled water (9 mL) was used]. After stirring for 30 minutes, anacquired solution was filtered with a syringe filter (Corning, 0.2 μm).After washing with DMSO wash liquid (0.5 mL), the wash liquid waswashed/filtered through a syringe filter and mixed. This solution wasdefined as a solution A.

The solution A (50 μL) was quickly mixed into NaCl aqueous solutions (50μL) of various concentrations to acquire dispersion liquid ofnanoparticles of αβ-DL-PolyAsp-D-PAE. The nanoparticles were measured interms of mean particle diameter [Z-Ave d. (nm)] and particle diameterdispersion index (PDI) by Zetasizer Nano ZS (Malvern). When the NaClaqueous solutions of various concentrations were used for preparation,the following results were acquired: 0.25 M NaCl, 554 nm (PDI 0.206);0.20 M NaCl, 214 nm (PDI 0.064); 0.15 M NaCl, 132 nm (PDI 0.030), 0.10 MNaCl, 87 nm (PDI 0.090); and 0.05 M NaCl, 69 nm (PDI 0.628).

The solution A (500 μL) was quickly mixed into 0.20 M NaCl aqueoussolution (500 μL) to acquire dispersion liquid (Z-Ave d., 72 nm, PDI0.149). The liquid was desalted by using Amicon Ultra 0.5 (Millipore, 10K) (centrifugation conditions: 4500 rpm, 15 minutes, 5° C.). Theacquired residue was subjected to lyophilization to acquirenanoparticles of αβ-DL-PolyAsp-D-PAE (7 mg, PAE introduction rate: 70%).

Example 99 Preparation of Nanoparticles of αβ-DL-PolyAsp-L-PAE

In a 5-mL sample bottle, αβ-DL-PolyAsp-L-PAE (50 mg, PAE introductionrate: 63%) and DMSO (0.5 mL) were measured and dissolved at roomtemperature. At room temperature, Na₂CO₃ aqueous solution was addeddropwise [0.15 mL of solution acquired by dissolving Na₂CO₃ (63 mg) indistilled water (9 mL) was used]. After stirring for 30 minutes, anacquired solution was filtered with a syringe filter (Corning, 0.2 μm).After washing with DMSO wash liquid (0.5 mL), the wash liquid waswashed/filtered through a syringe filter and mixed. This solution wasdefined as a solution A.

The solution A (50 μL) was quickly mixed into NaCl aqueous solutions (50μL) of various concentrations to acquire dispersion liquid ofnanoparticles of αβ-DL-PolyAsp-L-PAE. The nanoparticles were measured interms of mean particle diameter [Z-Ave d. (nm)] and particle diameterdispersion index (PDI) by Zetasizer Nano ZS (Malvern). When the NaClaqueous solutions of various concentrations were used for preparation,the following results were acquired: 0.25 M NaCl, 418 nm (PDI 0.151);0.20 M NaCl, 236 nm (PDI 0.199); 0.15 M NaCl, 126 nm (PDI 0.108), 0.10 MNaCl, 73 nm (PDI 0.090); and 0.05 M NaCl, 57 nm (PDI 0.140).

The solution A (500 μL) was quickly mixed into 0.20 M NaCl aqueoussolution (500 μL) to acquire dispersion liquid (Z-Ave d., 66 nm, PDI0.134). The liquid was desalted by using Amicon Ultra 0.5 (Millipore, 10K) (centrifugation conditions: 4500 rpm, 15 minutes, 5° C.). Theacquired residue was subjected to lyophilization to acquirenanoparticles of αβ-DL-PolyAsp-L-PAE (7 mg, PAE introduction rate: 71%).

As described in Examples, the present invention enables the acquisitionof the free form of the graft copolymer of a poly(amino acid) or a saltthereof and a hydrophobic primary amine compound or a salt thereof in ashort time at a high yield, and the ionized graft copolymer was able tobe acquired with a small amount of solvent (the sum of the solutions Aand B).

Example 100 Measurement of Absolute Molecular Weight of γ-PGA

The absolute molecular weights of γ-PGAs [12.1 g, 65 kDa, D:L ratio(35:65)] synthesized in accordance with Examples 5 and 6 were measuredby using GPC-Max (Viscotek) under SEC-HPLC conditions [TSKgelGMPWXL,300×7.8 mm I.D. (dual), 0.1 M NaNO₃, 0.8 mL/minute, 40° C.]. After eachof the γ-PGAs (129 mg) was dissolved in NaHCO₃ (0.50, 0.75, 1.00 1.25,and 1.50 equivalents relative to the total amount of carboxyl groups ofγ-PGA; 25 mL each), deaeration was conducted to acquire five γ-PGAsassociated with Na. Each of the γ-PGAs was filtered through a 0.20 μmfilter and used as a sample solution used with GPC-Max. From themolecular weights acquired form the five γ-PGAs associated with Na, theabsolute molecular weight value of free-body γ-PGA was calculated. Theabsolute molecular weights of Examples 5 and 6 were both 62 kDa.

The relative molecular weights of Examples 5 and 6 are both 65 kDa andthis indicates that correlation exists between the absolute molecularweight and the relative molecular weight of γ-PGA of the presentinvention.

Example 101 Synthesis of γ-PGA-PAE

In a 500-mL four-necked flask, distilled water (150 mL) and NaHCO₃ (0.66g) were measured at room temperature and dissolved. To this solution,γ-PGA (0.95 g, 148 kDa) was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (1.41 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (1.18 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. The solution was heated to 80° C. and 2 Mhydrochloric acid was added dropwise to adjust pH to 4.0. After stirringat 80° C. for 1 hour, the solution was cooled to room temperature andstirred for 2 hours. A precipitate was sucked and filtered and waswashed with distilled water (18 mL) twice. The precipitate was driedunder reduced pressure at room temperature to acquire γ-PGA-PAE (1.627g, yield: about 84.9%, PAE introduction rate: 61%, moisture: 3.9%, 91kDa).

Example 102 Synthesis of γ-PGA-PAE

In a 2-L four-necked flask, distilled water (950 mL) and NaHCO₃ (4.00 g)were measured at room temperature and dissolved. To this solution, γ-PGA(5.81 g, 148 kDa) was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (8.61 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (11.4 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. The solution was heated to 80° C. and 2 Mhydrochloric acid was added dropwise to adjust pH to 1.4. After stirringat 80° C. for 1 hour, the solution was cooled to room temperature andstirred for 2 hours. A precipitate was sucked and filtered and waswashed with distilled water (18 mL) twice. The precipitate was driedunder reduced pressure at room temperature to acquire γ-PGA-PAE (10.47g, yield: about 92.3%, PAE introduction rate: 69.1%, moisture: 3.4%, 100kDa).

Example 103 Synthesis of γ-PGA-PAE

In a 500-mL four-necked flask, distilled water (150 mL) and NaHCO₃ (0.66g) were measured at room temperature and dissolved. To this solution,γ-PGA (0.95 g, 148 kDa) was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (1.41 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (1.18 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. The solution was heated to 60° C. and 2 Mhydrochloric acid was added dropwise to adjust pH to 4.0. After stirringat 60° C. for 1 hour, the solution was cooled to room temperature andstirred for 2 hours. A precipitate was sucked and filtered and waswashed with distilled water (18 mL) twice. The precipitate was driedunder reduced pressure at room temperature to acquire γ-PGA-PAE (1.541g, yield: about 86.9%, PAE introduction rate: 60%, moisture: 2.4%, 160kDa).

Example 104 Synthesis of γ-PGA-PAE

In a 2-L four-necked flask, distilled water (950 mL) and NaHCO₃ (4.00 g)were measured at room temperature and dissolved. To this solution, γ-PGA(5.81 g, 148 kDa) was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (8.61 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (11.4 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. The solution was heated to 60° C. and 2 Mhydrochloric acid was added dropwise to adjust pH to 1.4. After stirringat 60° C. for 1 hour, the solution was cooled to room temperature andstirred for 2 hours. A precipitate was sucked and filtered and waswashed with distilled water (18 mL) twice. The precipitate was driedunder reduced pressure at room temperature to acquire γ-PGA-PAE (10.30g, yield: about 91.2%, PAE introduction rate: 69%, moisture: 3.6%, 211kDa).

Example 105 Synthesis of γ-PGA-PAE

In a 500-mL four-necked flask, distilled water (150 mL) and NaHCO₃ (0.66g) were measured at room temperature and dissolved. To this solution,γ-PGA (0.95 g, 148 kDa) was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (1.41 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (1.18 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. The solution was heated to 40° C. and 2 Mhydrochloric acid was added dropwise to adjust pH to 3.5. After stirringat 40° C. for 1 hour, the solution was cooled to room temperature andstirred for 2 hours. A precipitate was sucked and filtered and waswashed with distilled water (18 mL) twice. The precipitate was driedunder reduced pressure at room temperature to acquire γ-PGA-PAE (1.880g, yield: about 91.3%, PAE introduction rate: 60%, moisture: 4.3%, 203kDa).

Example 106 Synthesis of γ-PGA-PAE

In a 500-mL four-necked flask, distilled water (150 mL) and NaHCO₃ (0.66g) were measured at room temperature and dissolved. To this solution,γ-PGA (0.95 g, 148 kDa) was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (1.41 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (1.18 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. The solution was heated to 40° C. and 2 Mhydrochloric acid was added dropwise to adjust pH to 2.5. After stirringat 40° C. for 1 hour, the solution was cooled to room temperature andstirred for 2 hours. A precipitate was sucked and filtered and waswashed with distilled water (18 mL) twice. The precipitate was driedunder reduced pressure at room temperature to acquire γ-PGA-PAE (1.622g, yield: about 89.9%, PAE introduction rate: 61%, moisture: 3.0%, 220kDa).

Example 107 Synthesis of γ-PGA-PAE

In a 2-L four-necked flask, distilled water (950 mL) and NaHCO₃ (4.00 g)were measured at room temperature and dissolved. To this solution, γ-PGA(5.81 g, 148 kDa) was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (8.61 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (11.4 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. The solution was heated to 40° C. and 2 Mhydrochloric acid was added dropwise to adjust pH to 1.4. After stirringat 40° C. for 1 hour, the solution was cooled to room temperature andstirred for 2 hours. A precipitate was sucked and filtered and waswashed with distilled water (18 mL) twice. The precipitate was driedunder reduced pressure at room temperature to acquire γ-PGA-PAE (10.26g, yield: about 92.4%, PAE introduction rate: 69%, moisture: 3.1%, 235kDa).

Example 108 Synthesis of γ-PGA-PAE

In a 500-mL four-necked flask, distilled water (150 mL) and NaHCO₃ (0.63g) were measured at room temperature and dissolved. To this solution,γ-PGA (0.91 g, 148 kDa) was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (1.35 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (1.13 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. At room temperature, 2 M hydrochloric acid(33 mL) was added dropwise to adjust pH to 4.0. The solution was stirredfor 2 hours at room temperature. A precipitate was sucked and filteredand was washed with distilled water (18 mL) twice. The precipitate wasdried under reduced pressure at room temperature to acquire γ-PGA-PAE(1.596 g, yield: about 87.1%, PAE introduction rate: 61%, moisture:3.4%, 207 kDa).

Example 109 Synthesis of γ-PGA-PAE

In a 500-mL four-necked flask, distilled water (150 mL) and NaHCO₃ (0.63g) were measured at room temperature and dissolved. To this solution,γ-PGA (0.91 g, 148 kDa) was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (1.35 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (1.13 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. At room temperature, 2 M hydrochloric acid(3.6 mL) was added dropwise to adjust pH to 3.5. The solution wasstirred for 2 hours at room temperature. A precipitate was sucked andfiltered and was washed with distilled water (18 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-PAE (1.621 g, yield: about 90.5%, PAE introduction rate:61%, moisture: 3.5%, 208 kDa).

Example 110 Synthesis of γ-PGA-PAE

In a 500-mL four-necked flask, distilled water (150 mL) and NaHCO₃ (0.63g) were measured at room temperature and dissolved. To this solution,γ-PGA (0.91 g, 148 kDa) was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (1.35 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (1.13 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. At room temperature, 2 M hydrochloric acid(3.8 mL) was added dropwise to adjust pH to 3.0. The solution wasstirred for 2 hours at room temperature. A precipitate was sucked andfiltered and was washed with distilled water (18 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-PAE (1.543 g, yield: about 90.4%, PAE introduction rate:63%, moisture: 3.2%, 210 kDa).

Example 111 Synthesis of γ-PGA-PAE

In a 500-mL four-necked flask, distilled water (150 mL) and NaHCO₃ (0.63g) were measured at room temperature and dissolved. To this solution,γ-PGA (0.91 g, 148 kDa) was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (1.35 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (1.13 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. At room temperature, 2 M hydrochloric acid(4.4 mL) was added dropwise to adjust pH to 2.0. The solution wasstirred for 2 hours at room temperature. A precipitate was sucked andfiltered and was washed with distilled water (18 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-PAE (1.397 g, yield: about 90.4%, PAE introduction rate:61%, moisture: 3.1%, 205 kDa).

Example 112 Synthesis of γ-PGA-PAE

In a 500-mL four-necked flask, distilled water (150 mL) and NaHCO₃ (0.66g) were measured at room temperature and dissolved. To this solution,γ-PGA [0.95 g, 47 kDa, the absolute molecular weight was measured byGPC-Max (Viscotek)] was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (1.41 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (1.01 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. The solution was heated to 40° C. and 2 Mhydrochloric acid was added dropwise to adjust pH to 3.5. After stirringat 40° C. for 1 hour, the solution was cooled to room temperature andstirred for 2 hours. A precipitate was sucked and filtered and waswashed with distilled water (18 mL) twice. The precipitate was driedunder reduced pressure at room temperature to acquire γ-PGA-PAE (1.50 g,yield: about 92%, PAE introduction rate: 54%, moisture: 3.8%, 78 kDa).

Example 113 Synthesis of γ-PGA-PAE

In a 2-L four-necked flask, distilled water (950 mL) and NaHCO₃ (4.00 g)were measured at room temperature and dissolved. To this solution, γ-PGA(5.81 g, 148 kDa) was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (8.61 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (7.22 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. The solution was heated to 60° C. and 2 Mhydrochloric acid was added dropwise to adjust pH to 3.0. After stirringat 60° C. for 1 hour, the solution was cooled to room temperature andstirred for 2 hours. A precipitate was sucked and filtered and waswashed with distilled water (18 mL) twice. The precipitate was driedunder reduced pressure at room temperature to acquire γ-PGA-PAE (9.18 g,yield: about 88.8%, PAE introduction rate: 58%, moisture: 3.4%, 132kDa).

Example 114 Synthesis of γ-PGA-PAE

In a 2-L four-necked flask, distilled water (950 mL) and NaHCO₃ (4.00 g)were measured at room temperature and dissolved. To this solution, γ-PGA(5.81 g, 148 kDa) was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (8.61 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (7.22 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. The solution was heated to 80° C. and 2 Mhydrochloric acid was added dropwise to adjust pH to 3.0. After stirringat 80° C. for 1 hour, the solution was cooled to room temperature andstirred for 2 hours. A precipitate was sucked and filtered and waswashed with distilled water (18 mL) twice. The precipitate was driedunder reduced pressure at room temperature to acquire γ-PGA-PAE (9.77 g,yield: about 88.2%, PAE introduction rate: 57%, moisture: 3.6%, 86 kDa).

Example 115 Synthesis of γ-PGA-PAE

In a 2-L four-necked flask, distilled water (950 mL) and NaHCO₃ (4.00 g)were measured at room temperature and dissolved. To this solution, γ-PGA(5.81 g, 148 kDa) was added, stirred for 30 minutes under reducedpressure, and then ice-cooled. At ice temperature, WSC.HCl (8.61 g) wasadded and washed with distilled water (5 mL). After this solution wasstirred for 5 minutes, L-phenylalanine ethyl ester hydrochloride(PAE.HCl) (5.17 g) was added and washed with distilled water (5 mL). Thesolution was stirred for 1 hour at ice temperature and then stirred for20 hours at room temperature. The solution was heated to 80° C. and 2 Mhydrochloric acid was added dropwise to adjust pH to 3.0. After stirringat 80° C. for 1 hour, the solution was cooled to room temperature andstirred for 2 hours. A precipitate was sucked and filtered and waswashed with distilled water (18 mL) twice. The precipitate was driedunder reduced pressure at room temperature to acquire γ-PGA-PAE (8.25 g,yield: about 84.4%, PAE introduction rate: 44%, moisture: 4.3%, 55 kDa).

Example 116 Synthesis of γ-PGA-PAE

In a 100-mL Scott bottle, distilled water (35 mL) and 1M NaOH (9.0 mL)were measured at room temperature. To this solution, γ-PGA [1.21 g, 47kDa, the absolute molecular weight was measured by GPC-Max (Viscotek)]was added, dissolved with stirring for 15 minutes, and then ice-cooled.At ice temperature, WSC.HCl (1.80 g) was added and washed with distilledwater (5 mL). After this solution was stirred for 5 minutes,L-phenylalanine ethyl ester hydrochloride (PAE.HCl) (1.30 g) was addedand washed with distilled water (5 mL). The solution was stirred for 1hour at ice temperature and then stirred for 20 hours at roomtemperature. The solution was heated to 40° C. and 2 M hydrochloric acid(1.7 mL) was added dropwise to adjust pH to 3.5. The solution was cooledto room temperature and stirred for 1 hour. A precipitate was sucked andfiltered and was washed with distilled water (20 mL) twice. Theprecipitate was dried under reduced pressure at room temperature toacquire γ-PGA-PAE (2.1 g, yield: about 93%, PAE introduction rate: 62%,moisture: 3.3%).

Examples 117-1 to 117-5 Preparation of γ-PGA-PAE Na Salt

γ-PGA-PAE (1.00 g, PAE introduction rate: 57%) and distilled water (10mL) were measured. A 1 M NaOH aqueous solution was added dropwise withstirring at room temperature to adjust pH of each solution as describedin Table 1. Desalting using Amicon Ultra 15 (Millipore, 10 K) wasfollowed by washing with distilled water (5 mL). The desalting and thewashing operation were repeated thrice and an acquired solution wasfrozen and subjected to lyophilization to acquire each of γ-PGA-PAEsdescribed in Table 1.

TABLE 1 Na salt Yield PAE introduction Na Cl Examples pH (%) (g) rate(%) (ppm) (ppm) 117-1 3.0 0 0.785 58% 35 100 117-2 4.9 10 0.909 57%10000 100 117-3 7.4 23 0.920 57% 22000 100 117-4 9.0 26 0.923 59% 25000100 117-5 11 43 0.882 63% 40000 100

Examples 118-1 to 118-12 Preparation of γ-PGA-PAE Na Salt

γ-PGA-PAE (0.8 g, PAE introduction rate: 57%) and distilled water (8 mL)were measured. A 1.74 M Na₂CO₃ aqueous solution was added dropwise ineach amount described in Table 2 with stirring at room temperature.Desalting using Amicon Ultra 15 (Millipore, 10 K) was followed bywashing with distilled water (3 mL). The ultrafiltration and the washingoperation were repeated thrice and an acquired solution was frozen andsubjected to lyophilization to acquire each of γ-PGA-PAEs described inTable 2.

TABLE 2 Na₂CO₃ aqueous PAE solution Na salt Yield introduction Na ClExamples (mL) (%) (g) rate (%) (ppm) (ppm) 118-1 0.1 7 0.766 57 6500 180118-2 0.2 16 0.663 57 16000 100 118-3 0.3 23 0.728 59 22000 200 118-40.4 22 0.747 58 21000 200 118-5 0.5 22 0.767 58 21000 100 118-6 0.6 250.735 59 24000 150 118-7 0.7 27 0.793 59 26000 100 118-8 0.8 28 0.768 5927000 100 118-9 1.0 30 0.774 60 29000 100 118-10 1.2 31 0.770 61 29000100 118-11 1.4 30 0.782 62 28000 100 118-12 1.6 34 0.790 63 32000 100

Example 119 Preparation of Nanoparticles of γ-PGA-(Bn) Cys-OEt

γ-PGA-(Bn)Cys-OEt (100 mg, introduction rate: 60%, Example 42), DMSO(5.0 mL), and 100 mg/mL Na₂CO₃ aqueous solution (0.084 mL) were measuredand dissolved with stirring for 2 hours at room temperature to acquire aDMSO aqueous solution (concentration: 20 mg/mL) of γ-PGA-(Bn) Cys-OEt.An acquired solution was filtered with a syringe filter (Corning, 0.2μm) and this solution was defined as a solution A.

A portion of this solution A (concentration: 20 mg/mL, 0.05 mL) wasquickly mixed into NaCl aqueous solutions (0.05 mL) of variousconcentrations to acquire dispersion liquid. The liquid was desalted byusing Amicon Ultra 0:5 (Millipore, 10 K) (centrifugation conditions:4500 rpm, 30 minutes, 5° C.). An operation of dispersing an acquiredresidue in distilled water (0.10 mL) followed by desalting was performedtwice. The acquired residue was dispersed in 1×PBS (pH 7.4, 0.10 mL) toacquire a solution of nanoparticles of γ-PGA-(Bn)Cys-OEt (10 mg/mL). Thenanoparticles of γ-PGA-(Bn)Cys-OEt were measured in terms of meanparticle diameter [Z-Ave d. (nm)] and particle diameter dispersion index(PDI) by Zetasizer Nano ZS (Malvern). When the NaCl aqueous solutions ofvarious concentrations were used for preparation, the following resultswere acquired: 0.00 M NaCl; 33 nm (PDI 0.18); 0.10 M NaCl, 102 nm (PDI0.08); and 0.20 M NaCl, 211 nm (PDI 0.17).

NaCl (29.2 g) was dissolved in Otsuka distilled water (5 mL) andfiltered with a syringe filter (0.2 μm). This solution was defined as asolution B. A portion of the solution A (concentration: 20 mg/mL, 3.0mL) was mixed into a portion of the solution B [0.10 M NaCl aqueoussolution (3.0 mL)] to acquire dispersion liquid. The liquid was desaltedby using Amicon Ultra 15 (Millipore, 10K) (centrifugation conditions:4500 rpm, 30 minutes, 5° C.). An operation of dispersing an acquiredresidue in distilled water (6.0 mL) followed by desalting was performedfive times. Distilled water (10 mL) was added to the acquired residue toacquire a dispersion liquid C. A portion of the dispersion liquid C(0.05 mL) was dispersed in 1×PBS (1.0 mL) to acquire the dispersionliquid of nanoparticles (87 nm, PDI 0.09). The dispersion liquid C (4mL) was frozen in a freezer at −30° C. This frozen liquid was subjectedto lyophilization to acquire nanoparticles of γ-PGA-(Bn)Cys-OEt (15 mg).

Example 120 Preparation of Nanoparticles of γ-PGA-Trp-OEt

γ-PGA-Trp-OEt (100 mg, introduction rate: 56%, Example 43), DMSO (5.0mL), and 100 mg/mL Na₂CO₃ aqueous solution (0.128 mL) were measured anddissolved with stirring for 2 hours at room temperature to acquire aDMSO aqueous solution (concentration: 20 mg/mL) of γ-PGA-Trp-OEt. Anacquired solution was filtered with a syringe filter (Corning, 0.2 μm)and this solution was defined as a solution A.

A portion of this solution A (concentration: 20 mg/mL, 0.05 mL) wasquickly mixed into NaCl aqueous solutions (0.05 mL) of variousconcentrations to acquire dispersion liquid. The liquid was desalted byusing Amicon Ultra 0.5 (Millipore, 10 K) (centrifugation conditions:4500 rpm, 30 minutes, 5° C.). An operation of dispersing an acquiredresidue in distilled water (0.10 mL) followed by desalting was performedtwice. The acquired residue was dispersed in 1×PBS (pH 7.4, 0.10 mL) toacquire a solution of nanoparticles of γ-PGA-Trp-OEt (10 mg/mL). The thenanoparticles of γ-PGA-Trp-OEt were measured in terms of mean particlediameter [Z-Ave d. (nm)] and particle diameter dispersion index (PDI) byZetasizer Nano ZS (Malvern). When the NaCl aqueous solutions of variousconcentrations were used for preparation, the following results wereacquired: 0.00 M NaCl, 55 nm (PDI 0.12); 0.05 M NaCl, 92 nm (PDI 0.10);and 0.10 M NaCl, 153 nm (PDI 0.07).

NaCl (14.6 mg) was dissolved in Otsuka distilled water (5 mL) andfiltered with a syringe filter (0.2 μm). This solution was defined as asolution B. A portion of the solution A (concentration: 20 mg/mL, 3.0mL) was mixed into a portion of the solution B [0.05 M NaCl aqueoussolution (3.0 mL)] to acquire dispersion liquid. The liquid was desaltedby using Amicon Ultra 15 (Millipore, 10 K) (centrifugation conditions:4500 rpm, 30 minutes, 5° C.). An operation of dispersing an acquiredresidue in distilled water (6.0 mL) followed by desalting was performedfive times. Distilled water (10 mL) was added to the acquired residue toacquire a dispersion liquid C. A portion of the dispersion liquid C(0.05 mL) was dispersed in 1×PBS (1.0 mL) to acquire the dispersionliquid of nanoparticles (95 nm, PDI 0.13). The dispersion liquid C (4mL) was frozen in a freezer at −30° C. This frozen solution wassubjected to lyophilization to acquire nanoparticles of γ-PGA-Trp-OEt(16 mg).

Example 121 Preparation of Nanoparticles of γ-PGA-PAE

γ-PGA-PAE (200 mg, introduction rate: 62%, Example 116), DMSO (10.0 mL),and 100 mg/mL Na₂CO₃ aqueous solution (0.178 mL) were measured anddissolved with stirring for 2 hours at room temperature to acquire aDMSO aqueous solution (concentration: 20 mg/mL) of γ-PGA-PAE. Anacquired solution was filtered with a syringe filter (Corning, 0.2 μm)and this solution was defined as a solution A.

A portion of this solution A (concentration: 20 mg/mL, 0.05 mL) wasquickly mixed into NaCl aqueous solutions (0.05 mL) of variousconcentrations to acquire dispersion liquid. The liquid was desalted byusing Amicon Ultra 0.5 (Millipore, 10 K) (centrifugation conditions:4500 rpm, 30 minutes, 5° C.). An operation of dispersing an acquiredresidue in distilled water (0.10 mL) followed by desalting was performedtwice. The acquired residue was dispersed in 1×PBS (pH 7.4, 0.10 mL) toacquire a solution of nanoparticles of γ-PGA-PAE (10 mg/mL). Thenanoparticles of γ-PGA-PAE were measured in terms of mean particlediameter [Z-Ave d. (nm)] and particle diameter dispersion index (PDI) byZetasizer Nano ZS (Malvern). When the NaCl aqueous solutions of variousconcentrations were used for preparation, the following results wereacquired: 0.00 M NaCl, 56 nm (PDI 0.13); 0.05 M NaCl, 73 nm (PDI 0.12);0.06 M NaCl, 80 nm (PDI 0.10); 0.08 M NaCl, 91 nm (PDI 0.10); and 0.10 MNaCl, 124 nm (PDI 0.10).

NaCl (32.7 mg) was dissolved in Otsuka distilled water (7.0 mL) andfiltered with a syringe filter (0.2 μm). This solution was defined as asolution B. A portion of the solution A (concentration: 20 mg/mL, 4.5mL) was mixed into a portion of the solution B [0.08 M NaCl aqueoussolution (4.5 mL)] to acquire dispersion liquid. The liquid was desaltedby using Amicon Ultra 15 (Millipore, 10 K) (centrifugation conditions:4500 rpm, 30 minutes, 5° C.). An operation of dispersing an acquiredresidue in distilled water (10.0 mL) followed by desalting wasperformed, five times. Distilled water (15 mL) was added to the acquiredresidue to acquire a dispersion liquid C. A portion of the dispersionliquid C (0.05 mL) was dispersed in 1×PBS (1.0 mL) to acquire thedispersion liquid of nanoparticles (75 nm, PDI 0.11). The dispersionliquid C (6.0 mL) was frozen in a freezer at −30° C. This frozensolution was subjected to lyophilization to acquire nanoparticles ofγ-PGA-PAE (31 mg, PAE introduction rate: 59%, moisture: 3.4%).

Production of Vaccine

Example 122 Preparation of Liquid Mixture of Nanoparticles of γ-PGA-PAEand HA Antigen

The nanoparticles of γ-PGA-PAE (84.3 mg, PAE introduction rate: 57%,Example 77), 10×PBS (pH 7.4, 0.744 mL), and Otsuka distilled water(6.698 mL) were measured and stirred for 1 hour to acquire a dispersionliquid of nanoparticles of γ-PGA-PAE (concentration: 10 mg/mL). Aportion of the acquired solution (2.0 mL) was diluted with 1×PBS (8.0mL) to acquire a dispersion liquid of nanoparticles of γ-PGA-PAE (2mg/mL) (79 nm, PDI 0.27).

The acquired dispersion liquid (0.75 mL) was sequentially mixed with10×PBS (pH 7.4, 0.225 mL), distilled water (1.975 mL), and acommercially available HA antigen [Denka Seiken, Influenza HA Vaccine“SEIKEN”: active ingredients include the following three strains(A/California/7/2009 (H1N1) pdm09 strain; A/Texas/50/2012 (H3N2) strain;B/Massachusetts/2/2012 strain), the strains have HA contents(equivalents) of 30 μg or more/mL per strain, HA antigen: 1.5 μg ormore; 0.05 mL at 30 μg or more/mL] to prepare the HA vaccine (3.0 mL).

Example 123 Preparation of Liquid Mixture of Nanoparticles of γ-PGA-PAEand HA Antigen

The dispersion liquid acquired as described above (0.75 mL) wassequentially mixed with 10×PBS (pH 7.4, 0.225 mL), distilled water (2.02mL), and a commercially available HA antigen [Denka Seiken, Influenza HAVaccine “SEIKEN”: active ingredients include the following three strains(A/California/7/2009 (H1N1) pdm09 strain; A/Texas/50/2012 (H3N2) strain;B/Massachusetts/2/2012 strain), the strains have HA contents(equivalents) of 30 μg or more/mL per strain, HA antigen: 0.15 μg ormore; 0.005 mL at 30 μg or more/mL] to prepare the HA vaccine (3.0 mL).

Example 124 Preparation of Liquid Mixture of Nanoparticles of γ-PGA-PAEand HA Antigen

The dispersion liquid of nanoparticles of γ-PGA-PAE (1 mL, 20 mg/mL, PAEintroduction rate: 57%, dispersed in Example 78) was diluted with 1×PBS(9.0 mL) to acquire a dispersion liquid of nanoparticles of γ-PGA-PAE (2mg/mL) (78 nm, PDI 0.22).

The acquired dispersion liquid (0.75 mL) was sequentially mixed with10×PBS (pH 7.4, 0.225 mL), distilled water (1.975 mL), and acommercially available HA antigen [Denka Seiken, Influenza HA Vaccine“SEIKEN”: active ingredients include the following three strains(A/California/7/2009 (H1N1) pdm09 strain; A/Texas/50/2012 (H3N2) strain;B/Massachusetts/2/2012 strain), the strains have HA contents(equivalents) of 30 μg or more/mL per strain, HA antigen: 1.5 μg ormore; 0.05 mL at 30 μg or more/mL] to prepare the HA vaccine (3.0 mL).

Example 125 Preparation of Liquid Mixture of Nanoparticles of γ-PGA-PAEand HA Antigen

The dispersion liquid acquired as described above (0.75 mL) wassequentially mixed with 10×PBS (pH 7.4, 0.225 mL), distilled water (2.02mL), and a commercially available HA antigen [Denka Seiken, Influenza HAVaccine “SEIKEN”: active ingredients include the following three strains(A/California/7/2009 (H1N1) pdm09 strain; A/Texas/50/2012 (H3N2) strain;B/Massachusetts/2/2012 strain), the strains have HA contents(equivalents) of 30 μg or more/mL per strain, HA antigen: 0.15 μg ormore; 0.005 mL at 30 μg or more/mL] to prepare the HA vaccine (3.0 mL).

Example 126 Preparation of Liquid Mixture of Nanoparticles of γ-PGA-PAEand HA Antigen

γ-PGA-PAE (150 mg, introduction rate: 60%, Example 105), DMSO (5.45 mL),and Na₂CO₃ aqueous solution (5.2 mg/0.55 mL) were measured and dissolvedwith stirring for 2 hours at room temperature to acquire a DMSO aqueoussolution (concentration: 25 mg/mL) of γ-PGA-PAE. An acquired solutionwas filtered with a syringe filter (Corning, 0.2 μm) and this solutionwas defined as a solution A.

A portion of this solution A (concentration: 25 mg/mL, 0.10 mL) wasquickly mixed into 0.05 M NaCl aqueous solution (0.10 mL) to acquiredispersion liquid. The liquid was desalted by using Amicon Ultra 0.5(Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes, 5°C.). An operation of dispersing an acquired residue in distilled water(0.20 mL) followed by desalting was performed twice. The acquiredresidue was dispersed in 1×PBS (0.25 mL) to acquire a solution ofnanoparticles of γ-PGA-PAE (10 mg/mL). The nanoparticles of γ-PGA-PAEwere measured in terms of mean particle diameter [Z-Ave d. (nm)] andparticle diameter dispersion index (PDI) by Zetasizer Nano ZS (Malvern).The results were 0.05 M NaCl, 185 nm (PDI 0.10).

NaCl (11.7 mg) was dissolved in Otsuka distilled water (4.0 mL) andfiltered with a syringe filter (0.2 μm). This solution was defined as asolution B. A portion of the solution A (concentration: 25 mg/mL, 2.6mL) was mixed into a portion of the solution B [0.05 M NaCl aqueoussolution (2.6 mL)] to acquire dispersion liquid. The liquid was desaltedby using Amicon Ultra 15 (Millipore, 10 K) (centrifugation conditions:4500 rpm, 30 minutes, 5° C.). An operation of dispersing an acquiredresidue in distilled water (10 mL) followed by desalting was performedfive times. Distilled water (12 mL) was added to the acquired residue toacquire a dispersion liquid C. A portion of the dispersion liquid C(0.05 mL) was dispersed in 1×PBS (1.0 mL) to acquire the dispersionliquid of nanoparticles (197 nm, PDI 0.10). The dispersion liquid C (3.0mL) was frozen in a freezer at −30° C. This frozen solution wassubjected to lyophilization to acquire nanoparticles of γ-PGA-PAE (6 mg,PAE introduction rate: 57%, moisture: 2.2%). The dispersion liquid C(0.765 mL) was sequentially mixed with 10×PBS (pH 7.4, 0.30 mL),distilled water (1.885 mL), and a commercially available HA antigen[Denka Seiken, Influenza HA Vaccine “SEIKEN”: active ingredients includethe following three strains (A/California/7/2009 (H1N1) pdm09 strain;A/Texas/50/2012 (H3N2) strain; B/Massachusetts/2/2012 strain), thestrains have HA contents (equivalents) of 30 μg or more/mL per strain,HA antigen: 1.5 μg or more; 0.05 mL at 30 μg or more/mL] to prepare theHA vaccine (3.0 mL).

Example 127 Preparation of Liquid Mixture of Nanoparticles of γ-PGA-PAEand HA Antigen

The dispersion liquid C acquired as described above (0.765 mL) wassequentially mixed with 10×PBS (pH 7.4, 0.30 mL), distilled water (1.930mL), and a commercially available HA antigen [Denka Seiken, Influenza HAVaccine “SEIKEN”: active ingredients include the following three strains(A/California/7/2009 (H1N1) pdm09 strain; A/Texas/50/2012 (H3N2) strain;B/Massachusetts/2/2012 strain), the strains have HA contents(equivalents) of 30 μg or more/mL per strain, HA antigen: 0.15 μg formore; 0.005 mL at 30 μg or more/mL] to prepare the HA vaccine (3.0 mL).

Example 128 Preparation of Liquid Mixture of Nanoparticles of γ-PGA-PAEand HA Antigen

γ-PGA-PAE (150 mg, PAE introduction rate: 54%, Example 112), DMSO (4.45mL), and Na₂CO₃ aqueous solution (6.0 mg/0.55 mL) were measured anddissolved with stirring for 2 hours at room temperature to acquire aDMSO aqueous solution (concentration: 30 mg/mL) of γ-PGA-PAE. Anacquired solution was filtered with a syringe filter (Corning, 0.2 μm)and this solution was defined as a solution A.

A portion of this solution A (concentration: 30 mg/mL, 0.10 mL) wasquickly mixed into distilled water (0.10 mL) to acquire dispersionliquid. The liquid was desalted by using Amicon Ultra 0.5 (Millipore, 10K) (centrifugation conditions: 4500 rpm, 30 minutes, 5° C.). Anoperation of dispersing an acquired residue in distilled water (0.20 mL)followed by desalting was performed twice. The acquired residue wasdispersed in 1×PBS (0.30 mL) to acquire a solution of nanoparticles ofγ-PGA-PAE (10 mg/mL). The nanoparticles of γ-PGA-PAE were measured interms of mean particle diameter [Z-Ave d. (nm)] and particle diameterdispersion index (PDI) by Zetasizer Nano ZS (Malvern). The results were0.00 M NaCl, 199 nm (PDI 0.11).

Otsuka distilled water (4.0 mL) was filtered with a syringe filter (0.2μm). This solution was defined as a solution B. A portion of thesolution A (concentration: 30 mg/mL, 2.2 mL) was mixed into a portion ofthe solution B [distilled water (2.2 mL)] to acquire dispersion liquid.The liquid was desalted by using Amicon Ultra 15 (Millipore, 10 K)(centrifugation conditions: 4500 rpm, 30 minutes, 5° C.). An operationof dispersing an acquired residue in distilled water (10 mL) followed bydesalting was performed five times. Distilled water (12 mL) was added tothe acquired residue to acquire a dispersion liquid C. A portion of thedispersion liquid C (0.05 mL) was dispersed in 1×PBS (1.0 mL) to acquirethe dispersion liquid of nanoparticles (138 nm, PDI 0.11). A portion ofthe dispersion liquid C (3.0 mL) was frozen in a freezer at −30° C. Thisfrozen solution was subjected to lyophilization to acquire nanoparticlesof γ-PGA-PAE (12 mg, PAE introduction rate: 53%, moisture: 3.8%). Aportion of the dispersion liquid C (0.39 mL) was sequentially mixed with10×PBS (pH 7.4, 0.30 mL), distilled water (2.26 mL), and a commerciallyavailable HA antigen [Denka Seiken, Influenza HA Vaccine “SEIKEN”, 1.5μg or more; 0.05 mL at 30 μg or more/mL] to prepare the HA vaccine (3.0mL).

Example 129 Preparation of Liquid Mixture of Nanoparticles of γ-PGA-PAEand HA Antigen

The dispersion liquid C acquired as described above (0.39 mL) wassequentially mixed with 10×PBS (pH 7.4, 0.30 mL), distilled water (2.305mL), and a commercially available HA antigen [Denka Seiken, Influenza HAVaccine “SEIKEN”: active ingredients include the following three strains(A/California/7/2009 (H1N1) pdm09 strain; A/Texas/50/2012 (H3N2) strain;B/Massachusetts/2/2012 strain), the strains have HA contents(equivalents) of 30 μg or more/mL per strain, HA antigen: 1.5 μg ormore; 0.005 mL at 30 μg or more/mL] to prepare the HA vaccine (3.0 mL).

Example 130 Preparation of Liquid Mixture of Nanoparticles of γ-PGA-PAEand HA Antigen

A portion of the dispersion liquid C of nanoparticles of γ-PGA-PAEacquired in Example 121 (0.301 mL) was sequentially mixed with 10×PBS(pH7.4, 0.30 mL), distilled water (2.394 mL), and a commerciallyavailable HA antigen [Denka Seiken, Influenza HA Vaccine “SEIKEN”:active ingredients include the following three strains(A/California/7/2009 (H1N1) pdm09 strain; A/Texas/50/2012 (H3N2) strain;B/Massachusetts/2/2012 strain), the strains have HA contents(equivalents) of 30 μg or more/mL per strain, 0.15 μg or more; 0.005 mLat 30 μg or more/mL] to prepare the HA vaccine (3.0 mL).

Example 131 Preparation of Liquid Mixture of Nanoparticles ofγ-PGA-D-PAE and HA Antigen

γ-PGA-PAE (250 mg, PAE introduction rate: 56%, Example 11), DMSO (5.25mL), and Na₂CO₃ aqueous solution (23.3 mg/mL; 1.0 mL) were measured anddissolved with stirring for 2 hours at room temperature to acquire aDMSO aqueous solution (concentration: 40 mg/mL) of γ-PGA-PAE. Anacquired solution was filtered with a syringe filter (Corning, 0.2 μm)and this solution was defined as a solution A.

A portion of this solution A (concentration: 40 mg/mL, 0.10 mL) wasquickly mixed into NaCl aqueous solutions (0.10 mL) of variousconcentrations to acquire dispersion liquid. The liquid was desalted byusing Amicon Ultra 0.5 (Millipore, 10 K) (centrifugation conditions:4500 rpm, 30 minutes, 5° C.). An operation of dispersing an acquiredresidue in distilled water (0.20 mL) followed by desalting was performedtwice. The acquired residue was dispersed in 1×PBS (0.4 mL) to acquire asolution of nanoparticles of γ-PGA-PAE (10 mg/mL). The nanoparticles ofγ-PGA-PAE were measured in terms of mean particle diameter [Z-Ave d.(nm)] and particle diameter dispersion index (PDI) by Zetasizer Nano ZS(Malvern). The results were 0.05 M NaCl, 173 nm (PDI 0.10); 0.10 M NaCl,293 nm (PDI 0.26); and 0.15 M NaCl, 385 nm (PDI 0.39).

NaCl (14.6 mg) was dissolved in Otsuka distilled water (5.0 mL) andfiltered with a syringe filter (0.2 μm). This solution was defined as asolution B. A portion of the solution A (concentration: 40 mg/mL, 3.0mL) was mixed into a portion of the solution B [distilled water (3.0mL)] to acquire dispersion liquid. The liquid was desalted by usingAmicon Ultra 15 (Millipore, 10 K) (centrifugation conditions: 4500 rpm,30 minutes, 5° C.). An operation of dispersing an acquired residue indistilled water (10 mL) followed by desalting was performed five times.Distilled water (23 mL) was added to the acquired residue to acquire adispersion liquid C. A portion of the dispersion liquid C (0.05 mL) wasdispersed in 1×PBS (1.0 mL) to acquire the dispersion liquid ofnanoparticles (128 nm, PDI 0.07). A portion of the dispersion liquid C(3.0 mL) was frozen in a freezer at −30° C. This frozen solution wassubjected to lyophilization to acquire nanoparticles of γ-PGA-PAE (11mg, PAE introduction rate: 55%, moisture: 3.0%). A portion of thedispersion liquid C (0.422 mL) was sequentially mixed with 10×PBS (pH7.4, 0.30 mL), distilled water (2.273 mL), and a commercially availableHA antigen [Denka Seiken, Influenza HA Vaccine “SEIKEN”: activeingredients include the following three strains (A/California/7/2009(H1N1) pdm09 strain; A/Texas/50/2012 (H3N2) strain;B/Massachusetts/2/2012 strain), the strains have HA contents(equivalents) of 30 μg or more/mL per strain, HA antigen: 1.5 μg ormore; 0.005 mL at 30 μg or more/mL] to prepare the HA vaccine (3.0 mL).

Example 132 Preparation of Liquid Mixture of Nanoparticles ofα-D-PGA-L-PAE and HA Antigen

α-PGA-PAE (150 mg, PAE introduction rate: 69%, Example 21), DMSO (11.9mL), and Na₂CO₃ aqueous solution (6.4 mg/0.6 mL) were measured anddissolved with stirring for 2 hours at room temperature to acquire aDMSO aqueous solution (concentration: 12 mg/mL) of α-PGA-PAE. Anacquired solution was filtered with a syringe filter (Corning, 0.2 μm)and this solution was defined as a solution A.

A portion of this solution A (concentration: 12 mg/mL, 0.10 mL) wasquickly mixed into NaCl aqueous solutions (0.10 mL) of variousconcentrations to acquire dispersion liquid. The liquid was desalted byusing Amicon Ultra 0.5 (Millipore, 10 K) (centrifugation conditions:4500 rpm, 30 minutes, 5° C.). An operation of dispersing an acquiredresidue in distilled water (0.20 mL) followed by desalting was performedtwice. The acquired residue was dispersed in 1×PBS (0.12 mL) to acquirea solution of nanoparticles of α-PGA-PAE (10 mg/mL). The nanoparticlesof α-PGA-PAE were measured in terms of mean particle diameter [Z-Ave d.(nm)] and particle diameter dispersion index (PDI) by Zetasizer Nano ZS(Malvern). The results were 0.05 M NaCl, 155 nm (PDI 0.16); and 0.06 MNaCl, 178 nm (PDI 0.18).

NaCl (24.5 mg) was dissolved in Otsuka distilled water (7.0 mL) andfiltered with a syringe filter (0.2 μm). This solution was defined as asolution B. A portion of the solution A (concentration: 12 mg/mL, 5.0mL) was mixed into a portion of the solution B [0.06 M NaCl (5.0 mL)] toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra15 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (10 mL) followed by desalting was performed five times. Distilledwater (11 mL) was added to the acquired residue to acquire a dispersionliquid C. A portion of the dispersion liquid C (0.05 mL) was dispersedin 1×PBS (1.0 mL) to acquire the dispersion liquid of nanoparticles (129nm, PDI 0.22). A portion of the dispersion liquid C (3.0 mL) was frozenin a freezer at −30° C. This frozen solution was subjected tolyophilization to acquire nanoparticles of α-PGA-PAE (13 mg, PAEintroduction rate: 66%, moisture: 1.7%). A portion of the dispersionliquid C (0.352 mL) was sequentially mixed with 10×PBS (pH 7.4, 0.30mL), distilled water (2.343 mL), and a commercially available HA antigen[Denka Seiken, Influenza HA Vaccine “SEIKEN”: active ingredients includethe following three strains (A/California/7/2009 (H1N1) pdm09 strain;A/Texas/50/2012 (H3N2) strain; B/Massachusetts/2/2012 strain), thestrains have HA contents (equivalents) of 30 μg or more/mL per strain,HA antigen: 0.15 μg or more; 0.005 mL at 30 μg/mL or more] to preparethe HA vaccine (3.0 mL).

Example 133 Preparation of Liquid Mixture of Nanoparticles ofα-L-PGA-L-PAE and HA Antigen

α-PGA-PAE (60 mg, PAE introduction rate: 69%, Example 19), DMSO (9.4mL), and Na₂CO₃ aqueous solution (6.3 mg/0.6 mL) were measured anddissolved with stirring for 2 hours at room temperature to acquire aDMSO aqueous solution (concentration: 6 mg/mL) of α-PGA-PAE. An acquiredsolution was filtered with a syringe filter (Corning, 0.2 μm) and thissolution was defined as a solution A.

A portion of this solution A (concentration: 6 mg/mL, 0.10 mL) wasquickly mixed into NaCl aqueous solutions (0.10 mL) of variousconcentrations to acquire dispersion liquid. The liquid was desalted byusing Amicon Ultra 0.5 (Millipore, 10 K) (centrifugation conditions:4500 rpm, 30 minutes, 5° C.). An operation of dispersing an acquiredresidue in distilled water (0.20 mL) followed by desalting was performedtwice. The acquired residue was dispersed in 1×PBS (0.06 mL) to acquirea solution of nanoparticles of α-PGA-PAE (10 mg/mL). The nanoparticlesof α-PGA-PAE were measured in terms of mean particle diameter [Z-Ave d.(nm)] and particle diameter dispersion index (PDI) by Zetasizer Nano ZS(Malvern). The results were 0.10 M NaCl, 86 nm (PDI 0.13); 0.12 M NaCl,119 nm (PDI 0.15); 0.14 M NaCl, 173 nm (PDI 0.16); and 0.15 M NaCl, 374nm (PDI 0.30).

NaCl (98.2 mg) was dissolved in Otsuka distilled water (12.0 mL) andfiltered with a syringe filter (0.2 μm). This solution was defined as asolution B. A portion of the solution A (concentration: 6 mg/mL, 9.0 mL)was mixed into a portion of the solution B [0.14 M NaCl (9.0 mL)] toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra15 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (20 mL) followed by desalting was performed five times. Distilledwater (9 mL) was added to the acquired residue to acquire a dispersionliquid C. A portion of the dispersion liquid C (0.05 mL) was dispersedin 1×PBS (1.0 mL) to acquire the dispersion liquid of nanoparticles (183nm, PDI 0.18). A portion of the dispersion liquid C (3.0 mL) was frozenin a freezer at −30° C. This frozen solution was subjected tolyophilization to acquire nanoparticles of α-PGA-PAE (6 mg, PAEintroduction rate: 57%, moisture: 2.4%). A portion of the dispersionliquid C (0.769 mL) was sequentially mixed with 10×PBS (pH 7.4, 0.30mL), distilled water (1.926 mL), and a commercially available HA antigen[Denka Seiken, Influenza HA Vaccine “SEIKEN”: active ingredients includethe following three strains (A/California/7/2009 (H1N1) pdm09 strain;A/Texas/50/2012 (H3N2) strain; B/Massachusetts/2/2012 strain), thestrains have HA contents (equivalents) of 30 μg or more/mL per strain,HA antigen: 0.15 μg or more; 0.005 mL at 30 μg or more/mL) to preparethe HA vaccine (3.0 mL).

Example 134 Preparation of Liquid Mixture of Nanoparticles of αβ-DL-PolyAsp-L-PAE and HA Antigen

αβDL-Poly Asp-L-PAE (100 mg, PAE introduction rate: 62%, Example 23),DMSO (9.6 mL), and Na₂CO₃ aqueous solution (14.1 mg/0.4 mL) weremeasured and dissolved with stirring for 2 hours at room temperature toacquire a DMSO aqueous solution (concentration: 10 mg/mL) of αβ-DL-PolyAsp-L-PAE. An acquired solution was filtered with a syringe filter(Corning, 0.2 μm) and this solution was defined as a solution A.

A portion of this solution A (concentration: 10 mg/mL, 0.05 mL) wasquickly mixed into NaCl aqueous solutions (0.05 mL) of variousconcentrations to acquire dispersion liquid. The liquid was desalted byusing Amicon Ultra 0.5 (Millipore, 10 K) (centrifugation conditions:4500 rpm, 30 minutes, 5° C.). An operation of dispersing an acquiredresidue in distilled water (0.10 mL) followed by desalting was performedtwice. The acquired residue was dispersed in 1×PBS (0.05 mL) to acquirea solution of nanoparticles of αβ-DL-Poly Asp-L-PAE (10 mg/mL). Thenanoparticles of αβ-DL-Poly Asp-L-PAE were measured in terms of meanparticle diameter [Z-Ave d. (nm)] and particle diameter dispersion index(PDI) by Zetasizer Nano ZS (Malvern). The results were 0.05 M NaCl, 86nm (PDI 0.13); 0.10 M NaCl, 111 nm (PDI 0.10); 0.15 M NaCl, 155 nm (PDI0.04); 0.18 M NaCl, 192 nm (PDI 0.20); and 0.20 M NaCl, 220 nm (PDI0.06).

NaCl (105.2 mg) was dissolved in Otsuka distilled water (10.0 mL) andfiltered with a syringe filter (0.2 μm). This solution was defined as asolution B. A portion of the solution A (concentration: 10 mg/mL, 7.0mL) was mixed into a portion of the solution B [0.18 M NaCl (7.0 mL)] toacquire dispersion liquid. The liquid was desalted by using Amicon Ultra15 (Millipore, 10 K) (centrifugation conditions: 4500 rpm, 30 minutes,5° C.). An operation of dispersing an acquired residue in distilledwater (20 mL) followed by desalting was performed five times. Distilledwater (12 mL) was added to the acquired residue to acquire a dispersionliquid C. A portion of the dispersion liquid C (0.05 mL) was dispersedin 1×PBS (1.0 mL) to acquire the dispersion liquid of nanoparticles (225nm, PDI 0.03). A portion of the dispersion liquid C (3.0 mL) was frozenin a freezer at −30° C. This frozen solution was subjected tolyophilization to acquire nanoparticles of αβ-DL-Poly Asp-L-PAE (14 mg,PAE introduction rate: 60%, moisture: 4.5%). A portion of the dispersionliquid C (0.337 mL) was sequentially mixed with 10×PBS (pH 7.4, 0.30mL), distilled water (2.358 mL), and a commercially available HA antigen[Denka Seiken, Influenza HA Vaccine “SEIKEN”: active ingredients includethe following three strains (A/California/7/2009 (H1N1) pdm09 strain;A/Texas/50/2012 (H3N2) strain; B/Massachusetts/2/2012 strain), thestrains have HA contents (equivalents) of 30 μg or more/mL per strain,HA antigen: 0.15 μg or more; 0.005 mL at 30 μg or more/mL) to preparethe HA vaccine (3.0 mL).

Example 135 Cellular Immunity Induction Experiment Using Liquid Mixtureof HA Antigen and Nanoparticles of γ-PGA-PAE

A. Materials

Materials were 0.05% tween 20-containing phosphate buffer solution(PBS-T), skim milk (Wako Pure Chemical Industries, 198-10605), BSA(Sigma, A7030), HRP-labeled anti-mouse IgG antibody (Abcam, Ab7068), TMBsubstrate solution (Sigma, T0440-100 mL), TMB stop solution (CST,7002L), wells (96-well flat-bottom plate, Nunc, 112372), HA antigens[commercially available from Denka Seiken, Influenza HA Vaccine“SEIKEN”: active ingredients include the following three strains(A/California/7/2009 (H1N1) pdm09 strain; A/Texas/50/2012 (H3N2) strain;B/Massachusetts/2/2012 strain), the strains have HA contents(equivalents) of 30 μg or more/mL per strain], aluminum hydroxide gel(sterilized) [Alum: 2.0 mg/mL, volume: 1.0 mL, pH 7.045, physiologicalsaline (0.85% NaCl)], and various γ-PGA-PAE NPs prepared in the methodsof Examples described above. Various vaccine solutions were PBS (200 μL)solutions of HA antigens (0.1 μg or 0.01 μg) and nanoparticles ofγ-PGA-PAE (equivalent to 100 μg). Mice (BALB/c female mice, 7 week-old,Charles River Laboratories Japan, Inc.) were used.

B. Method

After about 1 week of acclimation of the mice (BALB/c female mice, 7week-old), 200 μL of a test substance was subcutaneously administered tothe mice by using a 1-mL syringe twice at an interval of 4 weeks. Serumswere collected after 2 weeks from the last immunization. For collectingthe serums, whole blood taken from the heart under pentobarbitalanesthesia was allowed to stand overnight at normal temperature and thencentrifuged (3000 rpm, 20 minutes) and each of the serums was dividedinto two and frozen at −80° C. An antibody titer was evaluated by usingthe frozen serum.

Preparation of Test Substance

(1) Preparation of Test Substance 1

In a sterile cryogenic vial (Corning), Otsuka distilled water (4.5 mL)and ×10 PBS (pH 7.4, 0.50 mL) were measured and sufficiently stirred.This solution was filtered with a syringe filter (0.2 μm) to acquire asolution A. A PBS preparation solution (blank solution without HA) wasacquired by measuring 3.0 mL from this solution A.

(2) Preparation of Test Substance 2

In a sterile cryogenic vial (Corning), Otsuka distilled water [2650 μLfiltered with a syringe filter (0.2 μm)] and ×10 PBS [pH 7.4; 300 μLfiltered with a syringe filter (0.2 μm)] were measured and sufficientlystirred. This solution was mixed with a commercially available HAantigen [Denka Seiken, Influenza HA Vaccine “SEIKEN”: active ingredientsinclude the following three strains (A/California/7/2009 (H1N1) pdm09strain; A/Texas/50/2012 (H3N2) strain; B/Massachusetts/2/2012 strain),the strains have HA contents (equivalents) of 30 μg or more/mL perstrain: equivalent to 1.5 μg or more of HA content (equivalent) of eachstrain, i.e., 0.05 mL was used at 30 μg or more/mL] to prepare HAvaccine (3.0 mL) (description of “or more” is based on an attacheddocument). This vaccine has HA concentration of 0.5 μg/mL or more.

(3) Preparation of Test Substance 3

In a sterile cryogenic vial (Corning), Otsuka distilled water [2695 μLfiltered with a syringe filter (0.2 μm)] and ×10 PBS [pH 7.4; 300 μLfiltered with a syringe filter (0.2 μm)] were measured and sufficientlystirred. This solution was mixed with a commercially available HAantigen [Denka Seiken, Influenza HA Vaccine “SEIKEN”: active ingredientsinclude the following three strains (A/California/7/2009 (H1N1) pdm09strain; A/Texas/50/2012 (H3N2) strain; B/Massachusetts/2/2012 strain),the strains have HA contents (equivalents) of 30 μg or more/mL perstrain: equivalent to 0.15 μg or more of HA content (equivalent) of eachstrain, i.e., 0.005 mL was used at 30 μg or more/mL] to prepare HAvaccine (3.0 mL) (description of “or more” is based on an attacheddocument). This vaccine has HA concentration of 0.05 μg/mL or more.

(4) Preparation of Test Substance 4

In a sterile cryogenic vial (Corning), 0.375 mL of aluminum hydroxidegel (Alum) (sterilized) [Alum: 2.0 mg/mL, volume: 1.0 mL, pH 7.045,physiological saline (0.85% NaCl)] was measured, and Otsuka distilledwater (0.973 mL) and ×10 PBS (pH 7.4, 0.15 mL) were then measured andsufficiently stirred. This solution was sequentially mixed with acommercially available HA antigen [Denka Seiken, Influenza HA Vaccine“SEIKEN”: active ingredients include the following three strains(A/California/7/2009 (H1N1) pdm09 strain; A/Texas/50/2012 (H3N2) strain;B/Massachusetts/2/2012 strain), the strains have HA contents(equivalents) of 30 μg or more/mL per strain: equivalent to 0.075 μg ormore of HA content (equivalent) of each strain, i.e., 0.0025 mL was usedat 30 μg or more/mL] to prepare control HA vaccine (1.5 mL) (descriptionof “or more” is based on an attached document). This vaccine has HAconcentration of 0.05 μg/mL or more.

(5) Preparation of Test Substance 5

For Example 127 using nanoparticles of γ-PGA-PAE instead of Alum, HAvaccine was prepared (3.0 mL) in the same way as Test Substance 4. Thisvaccine has HA concentration of 0.05 μg/mL or more.

(6) Preparation of Test Substance 6

For Example 129 using nanoparticles of γ-PGA-PAE instead of Alum, HAvaccine was prepared (3.0 mL) in the same way as Test Substance 4. Thisvaccine has HA concentration of 0.05 μg/mL or more.

(7) Preparation of Test Substance 7

For γ-PGA-PAE of Example 130 using nanoparticles of γ-PGA-PAE instead ofAlum, HA vaccine was prepared (3.0 mL) in the same way as Test Substance4. This vaccine has HA concentration of 0.05 μg/mL or more.

(8) Preparation of Test Substance 8

For Example 129 using nanoparticles of γ-PGA-PAE instead of Alum, HAvaccine was prepared (3.0 mL) in the same way as Test Substance 4. Thisvaccine has HA concentration of 0.05 μg/mL or more.

C. Evaluation Method

A titer of monoclonal antibody (mAb) to HA protein was evaluated as anevaluation item.

The HA antigen (containing 0.025 μg or more; 100 μL of coating buffer)is added to a well, refrigerated overnight, and then washed with PBS-Tby a microplate washer six times. After block buffer (200 μL) was addedto this well and allowed to act at room temperature for 2 hours, thewell was washed in the same way with PBS-T six times. After [testsubstance solution (such as serum/blank, see Table 3), 100 μL] was addedto this well and allowed to act at room temperature for 2 hours, thewell was washed in the same way with PBS-T six times. After ananti-mouse IgG antibody (100 μL) was added to this wall and allowed toact at room temperature for 2 hours, the well was washed in the same waywith PBS-T six times. After the TMB substrate solution (100 μL) wasadded to the well and allowed to act at room temperature for 10 minutes,the TMB stop solution (100 μL) was added. The acquired solution wasmeasured by a plate reader in terms of absorbance at 450 nm.

HA Antibody Administered content titer Group substance Details (μg/mL)[average] 1 Test PBS (blank) 0 13.0 substance 1 2 Test HA (x10) 0.5 or26.9 substance 2 more 3 Test HA (x1) 0.05 or 20.5 substance 3 more 4Test HA (x1) + Alum 0.05 or 25.7 substance 4 more 5 Test HA (x1) + 0.05or 26.7 substance 5 γ-PGA-PAE more Nanoparticles (Example 127) 6 Test HA(x1) + 0.05 or 26.9 substance 6 γ-PGA-PAE more Nanoparticles (Example129) 7 Test HA (x1) + 0.05 or 27.3 substance 7 γ-PGA-PAE moreNanoparticles (Example 130) 8 Test HA (x1) + 0.05 or 27.8 substance 8γ-PGA-PAE more Nanoparticles (Example 125)D. Results

These results (see Table 3 and FIG. 1) showed that the nanoparticles ofγ-PGA-PAE produced and prepared in the present invention have extremelyexcellent performance as an adjuvant.

Example 136 Physical Properties of γ-PGA-PAE Na Salt

The following table describes the relative molecular weight, the valuesof x, y, z in monomer units, and the total number (n) of monomer unitsin the graft copolymer of the γ-PGA-PAE Na salt synthesized as describedabove.

The relative molecular weight in the table was determined by a molecularweight measurement method using SEC-HPLC measurement: TSKgel α-M 300×7.8mm I.D. (dual), 5 mM NaNO₃ DMSO:H₂O (9:1), 0.8 mL/minute, 40° C., RIdetector, standard: pullulan (Shodex).

The values of x, y, and z in the table are determined by using thefollowing experiment method and the following calculation equationsrepresented by (Eq. 1) and (Eq. 2). By way of example, the calculationequations for γ-PGA-PAE (Na salt) are shown:

-   x: calculated from Eq. 2 after determination of [y] and [z]    described below;-   y: calculated from atomic absorption spectrometry or quantification    of Na ions by LCMS;-   z: calculated by ¹H-NMR as an introduction rate of PAE;-   molecular weight of γ-PGA-PAE (Na salt): calculated by a relative    molecular weight determination method using SEC-HPLC or an absolute    molecular weight determination method using viscometer, DLS    detector, SLS detector, and the like by GPC-Max (Viscotek) etc.; and-   n: the number of glutamic acid monomers in the graft copolymer (Glu    number).    [Math. 4]    Molecular weight of γ-PGA-PAE (Na salt)=[Molecular weight of    hydrogen of N terminal]+[(Molecular weight of    -Glu-OH—)×([n]×[x])]+[(Molecular weight of    -Glu-Ona-)×([n]×[y])]+[(Molecular weight of    -Glu-Phe-OEt-)×([n]×[z])]+[Molecular weight of hydroxyl group (or    sodium salt thereof) of C    terminal]=[1.007948×1]+[(12.0111×5+1.007947)×7+15.99943×3+14.00675×1+22.98977×0]×([n]×[x])+[(12.0111×5+1.007947×6+15.99943×3+14.00675×1+22.98977×1)×([n]×[y])]+[(12.0111×16+1.007947×20+15.99943×4+14.00675×2+22.98977×0)×([n]×[z])]+[15.99943×1+(1.007947×1    or 22.98977×1)]  (Eq. 1)    [Math. 5]    [x]+[y]+[z]=1  (Eq. 2)

The number of glutamic acid monomers in the graft copolymer (Glu number)was calculated from acquired experiment results by using the equationsimplemented as a spreadsheet of Excel 2010 (Microsoft). The results aredescribed in the following table.

The total number (n) of monomer units in the graft copolymer in thetable is the number derived from the number of glutamic acid monomers inthe graft copolymer (Glu number).

TABLE 4 Relative molecular Examples weight (kDa) x y z n 117-1 181 0.420.00 0.56 787 117-2 140 0.33 0.10 0.57 602 117-3 157 0.20 0.23 0.57 669117-4 108 0.15 0.26 0.59 452 117-5 61 0.15 0.43 0.63*¹ 244 118-1 1530.36 0.06 0.57 663 118-2 145 0.26 0.16 0.57 622 118-3 146 0.18 0.23 0.59616 118-4 163 0.20 0.22 0.58 690 118-5 152 0.20 0.22 0.58 646 118-6 1190.16 0.25 0.69 500 118-7 103 0.14 0.27 0.59 431 118-8 97 0.12 0.28 0.69403 118-9 80 0.10 0.30 0.69 335 118-10 69 0.09 0.31 0.61 283 118-11 760.09 0.30 0.62 311 118-12 60 0.03 0.34 0.63 241 *¹Ethyl ester of the PAEside chain was hydrolyzed by 21%.

Example 137 Calculation (Calculated Value) of Hydrophobic Parameter K (Clog P)

C log P values are calculated for the graft copolymers (γ-PGA-PAE) andthe ionized graft copolymers produced in accordance with Examplesdescribed above by ChemDraw Ultra (12.0.2.1076), Cambridgesoft. Theresults are described in the following table.

TABLE 5 Examples Clog P 1 −100 2 −264 3 −236 4 −38 5 31 6 −127 7 −48 8−2 10 −16 11 −106 12 −105 17 −1271 25 185 26 −61 27 360 28 −127 29 27330 54 31 −355 32 −532 33 −1675 34 −206 35 −233 36 252 37 195 39 277 40−158 41 −483 42 −519 43 600 44 −238 45 −163 46 −220 47 −215 48 −292 49−158 50 −216 51 −276 52 −307 117-1 −174 117-2 −181 117-3 −251 117-4 −170117-5 −101 118-1 −177 118-2 −206 118-3 −212 118-4 −243 118-5 −228 118-6−183 118-7 −162 118-8 −152 118-9 −131  118-10 −106  118-11 −110  118-12−87

Example 138 Measurement (Actual Measurement Value) of HydrophobicParameter K (log Pow)

γ-PGA-PAE [37 mg, n=403, x:y:z(12:28:59)] synthesized in accordance withExample 118-8 was suspended and stirred in 1-octanol (Wako SpecialGrade, 20 mL) and distilled water (20 mL) and allowed to standovernight. The solution was filtered by a 0.2-μm filter. Each of organicand water layers was sampled 5 mL by a transfer pipette and moderatelystirred for 1 hour and then allowed to stand still. Each of the organicand water layers was sampled 2 mL by a transfer pipette into a 25 mLmeasuring flask. To each of the solutions, 2 M sodium hydroxide aqueoussolution (10 mL) was added for hydrolysis at outside temperature of 50°C. for 2 hours. Each of the solutions was neutralized by adding 2 Mhydrochloric acid aqueous solution (10 mL) and then diluted in themeasuring flask. Phenylalanine in the sample (A) from the water layerand the sample (B) from the organic layer was quantitatively analyzed byHPLC (Sunniest RP-AQUA 100×2.0 mm i.d. 3 μm, UV 220 nm, 60° C., 0.4mL/minute; mobile phase A: 0.1% TFA aq.; mobile phase B: 0.1% TFA/MeCN;Program 0-5 minutes 5% B, 5-15 minutes 5-35% B, 15-18 minutes 35% B,18-23 minutes, 75% B; 23.01 minutes 5% B, Cycle: 30 minutes).

As a result, the phenylalanine content of (A) was (A) 0.012875 mg andthe phenylalanine content of (B) was 0.003700 mg. This results in logPow=log₁₀(0.003700/0.012875)=log₁₀(0.2874)=−0.542.

INDUSTRIAL APPLICABILITY

The present invention enables the acquisition of a free form of thegraft copolymer of a poly(amino acid) or a salt thereof and ahydrophobic primary amine compound or a salt thereof in a short time ata high yield, and the ionized graft copolymer may be acquired with asmall amount of solvent.

The acquired free form of the graft copolymer is hardly deliquesced andtherefore easily handled as a row material and makes quality controleasy since the state of the N terminal important for determination ofpolymer structure may be known.

When nanoparticles are formed from the free form of the graft copolymer,the hydrophobicity of the graft copolymer may be balanced by adjustingan ion species and an ionization amount to produce nanoparticlessuitable for a purpose.

Although concentration may be increased in the step of producingnanoparticles of the ionized graft copolymer of the present invention,the aggregation of acquired nanoparticles is suppressed and thereproducibility of forming/acquiring nanoparticles suitable for apurpose is increased by adjusting an ionization amount.

The invention claimed is:
 1. An ionized graft copolymer represented byFormula (II):

suitable for production of nanoparticles, wherein M is alkali metal, Ais a hydrophobic moiety, and n is an integer from 10 to 100,000;diagonal lines intervening three monomer units represent that themonomer units are arranged in irregular order, and x is a mole fractionof the monomer unit represented by Formula (III):

y is a mole fraction of the monomer unit represented by Formula (IV):

z is a mole fraction of the monomer unit represented by Formula (V):

and x, y, and z satisfy the following equations:0≤x<1;0<y<0.05;0<z<1; andx+y+z=1.
 2. The ionized graft copolymer according to claim 1, whereinthe mole fraction x of the monomer unit represented by Formula (III) is0.
 3. The ionized graft copolymer according to claim 1, wherein n is 50to 10,000.
 4. The ionized graft copolymer according to claim 1, whereinthe ionized graft copolymer has a hydrophobic parameter K of −15000 to0.
 5. A production method of an ionized graft copolymer, the methodcomprising a step of: ionizing a graft copolymer of a poly(amino acid)and a hydrophobic primary amine compound by allowing a hydroxide ofalkali metal, a carbonate of alkali metal, a hydrogencarbonate of alkalimetal, a phosphate of alkali metal, a monohydrogen phosphate of alkalimetal, a dihydrogen phosphate of alkali metal, an organic acid salt ofalkali metal, or an acidic amino-acid salt of alkali metal to act on thegraft copolymer to adjust a mole fraction of monomer units of the graftcopolymer having COOH side chains which are ionized by reaction with thealkali metal reactant in the poly(amino acid) to be more than 0.05 toless than 0.5, wherein the poly(amino acid) is selected from the groupconsisting of poly(γ-glutamic acid), poly(α-glutamic acid), andpoly(aspartic acid); and the hydrophobic primary amine compound isrepresented by Formula (I): A-NH₂, wherein A denotes a hydrophobicmoiety.
 6. A production method of nanoparticles containing an ionizedgraft copolymer of a poly(amino acid) selected from the group consistingof poly(γ-glutamic acid), poly(α-glutamic acid), and poly(asparticacid), or a salt thereof, and a hydrophobic primary amine compoundrepresented by Formula (I): A-NH₂ or a salt thereof, wherein A denotes ahydrophobic moiety, the method comprising the step of formingnanoparticles of the ionized graft copolymer produced with theproduction method according to claim
 5. 7. Nanoparticles produced by themethod according to claim 5, the nanoparticles containing an ionizedgraft copolymer of a poly(amino acid) selected from the group consistingof poly(γ-glutamic acid), poly(α-glutamic acid), and poly(asparticacid), or a salt thereof, and a hydrophobic primary amine compoundrepresented by Formula (I): A-NH₂ or a salt thereof, wherein A denotes ahydrophobic moiety.
 8. The nanoparticles according to claim 7, whereinthe nanoparticles are used as an adjuvant.
 9. A vaccine containing thenanoparticles according to claim 7.